Intraocular lymphoma detection reagent and detection method

Abstract

The present application relates to the technical field of auxiliary diagnosis of intraocular lymphoma, and specifically discloses an intraocular lymphoma detection reagent and detection method. The detection reagent includes a first reagent, and the first reagent includes at least five of CD117 antibody, CD56 antibody, CD45 antibody, CD38 antibody, CD34 antibody, CD33 antibody, CD19 antibody, CD13 antibody, CD7 antibody, kappa antibody and lambda antibody. The detection method includes (1) sample preparation; (2) initial screening; (3) typing. The specific expression of CD molecules indicates a specific disease, which is suitable for auxiliary diagnosis of lymphoma in clinical practice, and the detection result has high accuracy. According to the detection result of the first reagent, a preliminary screening and judgment can be carried out to confirm the major types of lymphoma.

Description

Technical Field

[0001] The present application relates to the field of intraocular lymphoma auxiliary diagnosis technology, and more specifically, it relates to an intraocular lymphoma detection reagent and detection method. Background Art

[0002] Intraocular lymphoma is usually divided into primary vitreoretinal lymphoma, primary choroidal lymphoma and intraocular metastasis of systemic lymphoma. It is a malignant tumor with relatively complex diagnosis. The complexity of diagnosis is mainly reflected in the small amount of intraocular fluid samples. Routine pathological examinations often encounter insufficient sample volume, but pathology is the gold standard for the diagnosis of lymphoma.

[0003] Flow cytometry immunophenotyping is a useful supplement to pathology. It detects multiple cell parameters at the same time, including cell number, size, surface antigens, etc. It can be used for auxiliary diagnosis in the diagnosis of intraocular lymphoma. The main advantage of the technology is that it can analyze trace amounts of cells.

[0004] Some highly suspected patients need to undergo repeated intraocular fluid testing to measure the levels of interleukins IL-10 and IL-6 in the intraocular fluid, which has a certain suggestive effect on the diagnosis of B-cell-derived primary vitreoretinal lymphoma. If the ratio of IL-10 to IL-6 is greater than 1, it indicates the possibility of lymphoma, and further related tests should be performed to confirm the diagnosis.

[0005] Although intraocular lymphoma is mainly derived from B cells, a small number of them are derived from T cells or NK cells. There is no literature proving that IL-10 concentration is related to lymphomas of T cells and other sources. In addition, there are patients with intraocular B cell-derived lymphoma whose IL-10 concentration is not detected to be elevated. Therefore, the determination of IL-10 and IL-6 content has a certain suggestive effect on the diagnosis of B cell-derived primary vitreoretinal lymphoma, but cannot assist in the diagnosis of T cell-derived lymphoma and NK cell-derived lymphoma. Summary of the invention

[0006] In order to improve the accuracy of auxiliary diagnosis of intraocular lymphoma, the present application provides an intraocular lymphoma detection reagent and detection method.

[0007] In a first aspect, the present application provides an intraocular lymphoma detection reagent, which adopts the following technical solution:

[0008] A reagent for detecting intraocular lymphoma comprises a first reagent, wherein the first reagent comprises at least five of CD117 antibody, CD56 antibody, CD45 antibody, CD38 antibody, CD34 antibody, CD33 antibody, CD19 antibody, CD13 antibody, CD7 antibody, kappa antibody and lambda antibody.

[0009] By adopting the above technical solution, the specific expression of CD molecules indicates a specific disease, which is suitable for clinical auxiliary diagnosis of lymphoma, and the test results are highly accurate. According to the test results of the first reagent, preliminary screening and judgment can be carried out to confirm the major types of lymphoma.

[0010] The kappa / lambda ratio is used to distinguish malignant lymphoma from benign lymphoma.

[0011] Abnormal expression of CD56: ① NK large granular lymphocytic leukemia, occasionally expressed in T-cell large granular lymphocytic leukemia; ② primitive NK cell leukemia / lymphoma; progressive NK cell leukemia / lymphoma; ③ nasal type NK cell lymphoma; ④ many acute myeloid leukemias; ⑤ can be expressed in multiple myeloma, sometimes in plasma cell leukemia; ⑥ some lung and nerve-related tumors (such as neuroblastoma, astrocytoma); ⑦ CD56 expression is associated with poor prognosis of AML with t(8;21) and t(15;17).

[0012] Abnormal expression of CD33: Most acute myeloid leukemias, especially M3 and M5, tend to show high expression; it may be weakly expressed in some acute lymphocytic leukemias.

[0013] Abnormal expression of CD13: Most of the primitive cells of acute myeloid leukemia, M6 and M7 may express negatively; some myeloma cells.

[0014] Abnormal expression of CD117: Most acute myeloid leukemia blasts; systemic mastocytosis; some myeloma cells (except plasma cell leukemia); rarely, it can be expressed in acute T-lymphocytic leukemia.

[0015] Abnormal expression of CD34: most acute myeloid leukemias; most acute B-lymphocytic leukemias (Pro-B or Common B, but pre-B and mature B do not express CD34); some acute T-lymphocytic leukemias.

[0016] Abnormal expression of CD7: 1. The original cells of acute T-lymphocytic leukemia; 2. T-lineage prolymphocytic leukemia cells; 3. T-lineage leukemia / lymphoma tumor cells; 4. Some acute myeloid leukemias. In this case, the expression of CD7 is helpful for monitoring the residual lesions of AML.

[0017] Abnormal expression of CD19: expressed in primitive cells of acute T-cell leukemia and tumor cells of mature T-cell leukemia / lymphoma.

[0018] Abnormal expression of CD38: myeloma cells, primary effusion lymphoma, some splenic lymphomas with villous lymphocytes, some chronic lymphocytic leukemias.

[0019] Abnormal expression of CD45: 1. Most acute T-lymphocytic leukemias express high levels of CD45, while 20% of acute B-lymphocytic leukemias are negative. 2. Acute myeloid leukemia weakly expresses CD45. 3. Neoplastic mast cells. 4. Both Hodgkin's cells and Reese's cells are CD45 negative, but Hodgkin's disease with nodular lymphocytes as the main type is CD45 positive. 5. It can occasionally be expressed in myeloma cells.

[0020] Optionally, the first reagent includes CD117 antibody, CD56 antibody, CD45 antibody, CD38 antibody, CD34 antibody, CD33 antibody, CD19 antibody, CD13 antibody, CD7 antibody, kappa antibody and lambda antibody.

[0021] By adopting the above technical solution, the screening of antibodies needs to take into account mutual interference and the synergy of expression prompt information. The combination of eleven antibodies in the first reagent can better achieve accurate screening of major lymphoma categories and provide a basis for subsequent auxiliary diagnosis of specific diseases.

[0022] For a 10-color flow cytometer, although only 10 channels of antibodies can be matched at a time, Kappa and CD56 have the same fluorescence and can use the same channel. Kappa and CD56 are expressed on different cells and do not interfere with each other. Therefore, the antibody combination of the first reagent can make full use of the channels of the flow cytometer, complete the initial screening at one time, and the accuracy of the initial screening results is high.

[0023] Optionally, a second reagent is also included, which includes CD16 antibody, CD22 antibody, CD4 antibody, CD5 antibody, CD23 antibody, CD20 antibody, CD3 antibody, CD138 antibody, CD19 antibody, CD8 antibody, CD45 antibody, CD300e antibody, CD64 antibody, CD123 antibody, CD85K antibody, CD14 antibody, CD36 antibody, CD2 antibody, CD11b antibody, CD303 antibody, CD71 antibody, CD304 antibody, CD10 antibody, MPO antibody, CD79a antibody, FMC7 antibody, IgM antibody, CD103 antibody, CD95 antibody, CD39 antibody, CD11c At least ten of the group consisting of antibody, CD30 antibody, CD25 antibody, CD40 antibody, CD43 antibody, BCL-2 antibody, CD200 antibody, ki-67 antibody, CD56 antibody, CD38 antibody, Kappa antibody, Lambda antibody, CD27 antibody, CD81 antibody, CD28 antibody, TdT antibody, CD9 antibody, CD66c antibody, HLA-DR antibody, CD307d antibody, CD1a antibody, CD57 antibody, CD45RA antibody, CD158e1 antibody, CD158e2 antibody, CD158b antibody, CD158i antibody, CD158f antibody, CD158a antibody and CD158h antibody.

[0024] Optionally, the second reagent comprises at least twenty antibodies.

[0025] By adopting the above technical solution, the first reagent and the second reagent are used successively for intraocular fluid sample detection, the antibody combination used by the second reagent is selected, and then the lymphoma is typed according to the detection of the second reagent, thereby further improving the accuracy.

[0026] Optionally, a third reagent is also included, and the third reagent includes a kit for detecting IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α in intraocular fluid.

[0027] By adopting the above technical solutions, some CD molecules that are not expressed obviously need to be analyzed in combination with cytokine conditions. An increase of more than 10 times in IL-10 alone and an increase in IFN-γ may indicate hemophilia, and EB virus infection and other possibilities need to be ruled out. IL-2, IFN-γ and TNF-α levels are significantly reduced, and a slight increase in IL-6 and IL-10 levels indicates possible progression of the tumor microenvironment. A significant increase in IL-4 may indicate an allergic reaction.

[0028] Optionally, each antibody in the first reagent is labeled with a fluorescent marker; and each antibody selected in the second reagent is labeled with a fluorescent marker.

[0029] In a second aspect, a method for detecting intraocular lymphoma, using an intraocular lymphoma detection reagent, comprises the following steps:

[0030] (1) Sample preparation:

[0031] Taking an intraocular fluid sample, centrifuging it, and removing the supernatant to obtain a concentrated sample;

[0032] (2) Initial screening:

[0033] The concentrated sample and the first reagent are mixed evenly, and then incubated in the dark, followed by washing with PBS buffer, centrifugation, removal of the supernatant, and then addition of PBS buffer for resuspending to obtain a test sample A;

[0034] The detection sample A is tested using a flow cytometer to obtain data.

[0035] By adopting the above technical scheme, after (2) initial screening, the major types of lymphoma, B type, T type or NK type, are confirmed.

[0036] Optionally, it also includes (3) typing, the specific process of which is as follows:

[0037] Determining the antibody combination selected for the second reagent based on the results of the initial screening of (2);

[0038] The concentrated sample and the second reagent were mixed evenly, and then incubated in the dark, followed by washing with PBS buffer, centrifugation continued, the supernatant was removed, and then PBS buffer was added and resuspended to obtain the test sample B;

[0039] Using flow cytometry to test sample B and obtain data;

[0040] The second reagent includes CD16 antibody, CD22 antibody, CD4 antibody, CD5 antibody, CD23 antibody, CD20 antibody, CD3 antibody, CD138 antibody, CD19 antibody, CD8 antibody, CD45 antibody, CD300e antibody, CD64 antibody, CD123 antibody, CD85K antibody, CD14 antibody, CD36 antibody, CD2 antibody, CD11b antibody, CD303 antibody, CD71 antibody, CD304 antibody, CD10 antibody, MPO antibody, CD79a antibody, FMC7 antibody, IgM antibody, CD103 antibody, CD95 antibody, CD39 antibody, CD11c antibody, CD30 At least twenty of the group consisting of antibodies, CD25 antibodies, CD40 antibodies, CD43 antibodies, BCL-2 antibodies, CD200 antibodies, ki-67 antibodies, CD56 antibodies, CD38 antibodies, Kappa antibodies, Lambda antibodies, CD27 antibodies, CD81 antibodies, CD28 antibodies, TdT antibodies, CD9 antibodies, CD66c antibodies, HLA-DR antibodies, CD307d antibodies, CD1a antibodies, CD57 antibodies, CD45RA antibodies, CD158e1 antibodies, CD158e2 antibodies, CD158b antibodies, CD158i antibodies, CD158f antibodies, CD158a antibodies, and CD158h antibodies.

[0041] By adopting the above technical solution, after (3) classification, the specific disease can be assisted in diagnosis.

[0042] Optionally, (3) typing also includes the following steps:

[0043] Take the third reagent, mix the cytokine microspheres and microsphere buffer, and incubate;

[0044] Then, the concentrated sample and antibody were added, mixed evenly, incubated, and then PBS buffer was added for washing, centrifuged, the supernatant was removed, and then PBS buffer was added for resuspending to obtain the test sample C;

[0045] Using flow cytometry to test sample C and obtain data;

[0046] The third reagent includes reagents for detecting IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α in intraocular fluid.

[0047] By adopting the above technical scheme, some CD molecules with unclear expression should be analyzed in combination with cytokine status.

[0048] Optionally, the intraocular fluid sample is aqueous humor or vitreous humor.

[0049] In summary, this application has the following beneficial effects:

[0050] 1. This application screens out a specific antibody composition as the first reagent to perform a preliminary screening of intraocular lymphoma, confirm the major types of lymphoma, and then conduct subsequent antibody / cytokine testing based on the preliminary screening results to classify the disease, providing a basis for clinical auxiliary diagnosis, with a high detection accuracy;

[0051] 2. This application uses flow cytometry as a detection method, which requires a small amount of sample and has high detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the first test result diagram of the application example of this application;

[0053] Figure 2 This is the second test result diagram of the application example of this application. DETAILED DESCRIPTION

[0054] The present application is further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can all be sourced from common commercial sources.

[0055] The embodiment of the present application discloses an intraocular lymphoma detection reagent, which includes a first reagent, a second reagent and a third reagent.

[0056] The first reagent includes at least five of CD117 antibody, CD56 antibody, CD45 antibody, CD38 antibody, CD34 antibody, CD33 antibody, CD19 antibody, CD13 antibody, CD7 antibody, kappa antibody and lambda antibody, preferably all of them are used. Each antibody in the first reagent is labeled with a different fluorescent marker, wherein only Kappa and CD56 are labeled with the same fluorescent marker.

[0057] The second reagent includes CD16 antibody, CD22 antibody, CD4 antibody, CD5 antibody, CD23 antibody, CD20 antibody, CD3 antibody, CD138 antibody, CD19 antibody, CD8 antibody, CD45 antibody, CD300e antibody, CD64 antibody, CD123 antibody, CD85K antibody, CD14 antibody, CD36 antibody, CD2 antibody, CD11b antibody, CD303 antibody, CD71 antibody, CD304 antibody, CD10 antibody, MPO antibody, CD79a antibody, FMC7 antibody, IgM antibody, CD103 antibody, CD95 antibody, CD39 antibody, CD11c antibody, CD30 antibody, CD 25 antibody, CD40 antibody, CD43 antibody, BCL-2 antibody, CD200 antibody, ki-67 antibody, CD56 antibody, CD38 antibody, Kappa antibody, Lambda antibody, CD27 antibody, CD81 antibody, CD28 antibody, TdT antibody, CD9 antibody, CD66c antibody, HLA-DR antibody, CD307d antibody, CD1a antibody, CD57 antibody, CD45RA antibody, CD158e1 antibody, CD158e2 antibody, CD158b antibody, CD158i antibody, CD158f antibody, CD158a antibody and CD158h antibody, at least ten, preferably at least twenty. The antibody selection of the second reagent is determined according to the preliminary screening results, and each antibody in the selected antibody combination is labeled with a different fluorescent marker, and two antibodies that do not interfere with each other can be labeled with the same fluorescent marker.

[0058] The fluorescent labels in the embodiments of the present application include but are not limited to the following types: AlexaFluor 488, ECD, PerCP, PerCP-cy5.5, PerCP-cy7, APC, Alexa Fluor 680, APC-Alexa Fluor700, APC-AlexaFluor750, APC-cy7, APC-H7, PacificBlue, Horizon V450, AmCyan, PE / Cyanine7, PE, FITC, Alexa Fluor ® 647, APC / Cyanine7 and Brilliant Violet 421 TM The fluorescent markers and antibodies with fluorescent markers can be purchased through commercial channels. Specifically, the fluorescent markers are selected as described in the embodiments.

[0059] The third reagent includes a kit for IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α. The kit is a human Th1 / Th2 subpopulation detection kit from Jiangxi Saiji Biotechnology Co., Ltd. The kit includes G sample diluent, H microsphere buffer, A capture microsphere mixture and fluorescent detection reagent. A capture microsphere mixture consists of polystyrene, anti-human IL-2 monoclonal antibody, anti-human IL-4 monoclonal antibody, anti-human IL-6 monoclonal antibody, anti-human IL-10 monoclonal antibody, anti-human IL-17A monoclonal antibody, anti-human TNF-α monoclonal antibody, and anti-human IFN-γ monoclonal antibody. The antibody manufacturer is Biolegend.

[0060] The PBS buffer in the examples of the present application is 1XPBS buffer.

[0061] The intraocular fluid sample in the embodiments of the present application is aqueous humor or vitreous humor. Specifically, vitreous humor is used in the embodiments of the present application.

[0062] The present application embodiment discloses a method for detecting intraocular lymphoma. The detection method comprises the following steps:

[0063] (1) Sample preparation:

[0064] The vitreous humor sample was collected and concentrated by centrifugation at 500 g, and the supernatant was removed, leaving 200 μl of the concentrated sample.

[0065] (2) Initial screening:

[0066] Take the flow tube number, add the antibody of the first reagent and premix, the addition amount of each antibody is 1.3μl-5μl, and it can be mixed in equal proportions or unequal proportions. In the embodiment of the present application, the addition amount is 2μl and mixed in equal proportions, and then add 50μl of the concentrated sample, vortex mix evenly, and then incubate in the dark for 15min, then add 1ml PBS buffer to wash, continue to centrifuge at 500g, remove the supernatant, and then add 300μl PBS buffer, resuspend, and obtain the test sample A;

[0067] The detection sample A is tested using a flow cytometer to obtain data.

[0068] (3) Classification:

[0069] Determining the antibody combination selected for the second reagent based on the results of the initial screening of (2);

[0070] Take the flow tube number, add the antibody of the second reagent and premix, the addition amount of each antibody is 1.3μl-5μl, and it can be mixed in equal proportions or unequal proportions. In the embodiment of the present application, the addition amount is 2μl and mixed in equal proportions, and then add 50μl of the concentrated sample, vortex mix evenly, and then incubate in the dark for 15min, then add 1ml PBS buffer to wash, continue to centrifuge at 500g, remove the supernatant, and then add 300μl PBS buffer, resuspend, and obtain the test sample B;

[0071] The detection sample B is tested using a flow cytometer to obtain data.

[0072] According to the results of the initial screening (2), if the expression of CD molecules is not obvious, the following steps are performed:

[0073] Mix the capture microsphere mixture and the microsphere buffer solution, the amount of the capture microsphere mixture added is 15-25 μl, in the embodiment of the present application, the amount of the capture microsphere mixture added is 20 μl, and the amount of the microsphere buffer added is 25 μl, and incubate for 30 minutes;

[0074] Then add 25 μl of the concentrated sample and 15-25 μl of the fluorescent detection reagent. In the embodiment of the present application, the amount of the fluorescent detection reagent added is 20 μl. Vortex to mix evenly, incubate for 2.5 hours, then add 1 ml of PBS buffer for washing, centrifuge at 500 g, remove the supernatant, and then add 200 μl of PBS buffer and resuspend to obtain a test sample C;

[0075] The detection sample C is tested using a flow cytometer to obtain data.

[0076] According to the results of the initial screening of (2), the following six situations are obtained: 1. Analysis is B cell abnormality; 2. Analysis is T cell / NK cell abnormality; 3. Analysis is plasma cell abnormality; 4. Analysis is B acute lymphoma leukemia; 5. Analysis is myeloid leukemia; 6. Analysis is normal. The second reagents selected for the above six situations refer to Examples 1-6.

[0077] Embodiment 1:

[0078] The first reagent includes CD117-APC, CD56-FITC, CD45-BV605, CD38-BV510, CD34-AlexaFluor700, CD33-PerCp-Cy5.5, CD19-BV421, CD13-PerCp-Cy7, CD7-APC-Cy7, Kappa-FITC and lambda-PE.

[0079] (2) The initial screening results showed B cell abnormalities.

[0080] The second reagent includes group one, group two and group three, and each group of antibodies is subjected to (3) typing test. The selection of each group of antibodies and fluorescent markers is shown in Table 1, where the horizontal direction of the table corresponds to the selection of fluorescent markers, and the vertical direction corresponds to the selection of each group of antibodies.

[0081] Table 1

[0082] A group Group 2 Three groups Fluorescent labeling 1 FMC7 CD103 / FITC 2 CD22 CD95 CD43 PE 3 CD5 CD39 / PerCp-Cy5.5 4 CD23 CD11c BCL-2 PerCp-Cy7 5 CD20 CD30 CD307d APC 6 slgM / / Alexa Fluor 700 7 CD138 CD25 CD19 APC-Cy7 8 CD19 CD19 CD200 BV421 9 CD10 CD40 ki-67 BV510 10 CD45 CD45 CD45 BV605

[0083] Embodiment 2:

[0084] The first reagent includes CD117-APC, CD56-FITC, CD45-BV605, CD38-BV510, CD34-AlexaFluor700, CD33-PerCp-Cy5.5, CD19-BV421, CD13-PerCp-Cy7, CD7-APC-Cy7, Kappa-FITC and lambda-PE.

[0085] (2) The initial screening results showed abnormalities in T cells and NK cells.

[0086] The second reagent includes group one, group two and group three, and each group of antibodies is subjected to (3) typing test. The selection of each group of antibodies and fluorescent markers is shown in Table 2, where the horizontal direction of the table corresponds to the selection of fluorescent markers, and the vertical direction corresponds to the selection of each group of antibodies.

[0087] Table 2

[0088] A group Group 2 Three groups Fluorescent labeling 1 CD56 CD56 CD56 FITC 2 CD4 / CD158d PE 3 CD5 CD158e1 / e2 / PerCp-Cy5.5 4 CDla CD158b / PerCp-Cy7 5 CD57 CD158i / APC 6 CD3 CD16 / Alexa Fluor 700 7 CD45RA CD158f / APC-Cy7 8 CD8 CD158a / h / BV421 9 CD10 / / BV510 10 CD45 CD45 CD45 BV605

[0089] Embodiment 3:

[0090] The first reagent includes CD117-APC, CD56-FITC, CD45-BV605, CD38-BV510, CD34-AlexaFluor700, CD33-PerCp-Cy5.5, CD19-BV421, CD13-PerCp-Cy7, CD7-APC-Cy7, Kappa-FITC and lambda-PE.

[0091] (2) The initial screening results showed abnormal plasma cells.

[0092] The second reagent includes group one and group two, and each group of antibodies is subjected to (3) typing test. The selection of each group of antibodies and fluorescent markers is shown in Table 3, where the horizontal direction of the table corresponds to the selection of fluorescent markers, and the vertical direction corresponds to the selection of each group of antibodies.

[0093] Table 3

[0094] A group Group 2 Fluorescent labeling 1 CD56 Kappa FITC 2 CD22 Lambda PE 3 CD5 CD27 PerCp-Cy5.5 4 CD23 BCL-2 PerCp-Cy7 5 CD20 CD81 APC 6 slgM clgM Alexa Fluor 700 7 CD138 CD38 APC-Cy7 8 CD200 CD28 BV421 9 CD38 ki-67 BV510 10 CD45 CD45 BV605

[0095] Embodiment 4:

[0096] The first reagent includes CD117-APC, CD56-FITC, CD45-BV605, CD38-BV510, CD34-AlexaFluor700, CD33-PerCp-Cy5.5, CD19-BV421, CD13-PerCp-Cy7, CD7-APC-Cy7, Kappa-FITC and lambda-PE.

[0097] (2) The initial screening results showed B acute lymphoma leukemia.

[0098] The second reagent includes group one and group two, and each group of antibodies is subjected to (3) typing test. The selection of each group of antibodies and fluorescent markers is shown in Table 4, where the horizontal direction of the table corresponds to the selection of fluorescent markers, and the vertical direction corresponds to the selection of each group of antibodies.

[0099] Table 4

[0100] A group Group 2 Fluorescent labeling 1 CD56 TdT FITC 2 CD22 CD22 PE 3 CD5 CD79a PerCp-Cy5.5 4 CD123 CD9 PerCp-Cy7 5 CD20 CD81 APC 6 slgM clgM Alexa Fluor 700 7 CD38 / APC-Cy7 8 CD200 CD66c BV421 9 CD10 / BV510 10 CD45 CD45 BV605

[0101] Embodiment 5:

[0102] The first reagent includes CD117-APC, CD56-FITC, CD45-BV605, CD38-BV510, CD34-AlexaFluor700, CD33-PerCp-Cy5.5, CD19-BV421, CD13-PerCp-Cy7, CD7-APC-Cy7, Kappa-FITC and lambda-PE.

[0103] (2) The initial screening results showed myeloid leukemia.

[0104] The second reagent includes group one, group two and group three, and each group of antibodies is subjected to (3) typing test. The selection of each group of antibodies and fluorescent markers is shown in Table 5, where the horizontal direction of the table corresponds to the selection of fluorescent markers, and the vertical direction corresponds to the selection of each group of antibodies.

[0105] Table 5

[0106] A group Group 2 Three groups Fluorescent labeling 1 CD16 CD16 MPO FITC 2 CD300e CD22 CD3 PE 3 CD64 CD11b CD79a PerCp-Cy5.5 4 CD123 CD303 / PerCp-Cy7 5 CD85k / / APC 6 CD14 / / Alexa Fluor 700 7 CD36 CD71 / APC-Cy7 8 CD2 HLA-DR CD304 BV421 9 CD4 CD10 / BV510 10 CD45 CD45 CD45 BV605

[0107] Embodiment 6:

[0108] The first reagent includes CD117-APC, CD56-FITC, CD45-BV605, CD38-BV510, CD34-AlexaFluor700, CD33-PerCp-Cy5.5, CD19-BV421, CD13-PerCp-Cy7, CD7-APC-Cy7, Kappa-FITC and lambda-PE.

[0109] (2) The initial screening results were normal.

[0110] The second reagent includes group one and group two, and each group of antibodies is subjected to (3) typing test. The selection of each group of antibodies and fluorescent markers is shown in Table 6, where the horizontal direction of the table corresponds to the selection of fluorescent markers, and the vertical direction corresponds to the selection of each group of antibodies.

[0111] Table 6

[0112] A group Group 2 Fluorescent labeling 1 CD56 CD16 FITC 2 CD4 CD22 PE 3 CD5 CD11b PerCp-Cy5.5 4 CD1a CD303 PerCp-Cy7 5 CD57 / APC 6 CD3 / Alexa Fluor 700 7 CD45RA CD71 APC-Cy7 8 CD8 HLA-DR BV421 9 CD10 CD10 BV510 10 CD45 CD45 BV605

[0113] Application examples:

[0114] The patient was a 70-year-old female who was diagnosed with decreased vision and bleeding. Vitreous mixing was found during the examination, and intraocular lymphoma was clinically considered. The intraocular lymphoma detection method of the embodiment of the present application was used for detection, and the detection instrument was BD FACS Canto IIPLUS (3 lasers and 10 colors).

[0115] Test results such as Figure 1 , Figure 2 As shown in the figure, (2) in the initial screening results, abnormal B cell populations accounted for 72.312% of the total. (3) In the typing results, CD19+ indicates lymphoma; CD20++ indicates B cell lymphoma; CD22+ indicates B cell lymphoma; CD38+ indicates normal early B and germinal center B cells, but the result shows CD38-, indicating B cell abnormality; CD5+ indicates normal B cells, but the result shows CD5-, indicating B cell abnormality; CD10+ indicates normal early B cells, germinal center B cells and non-neoplastic immature B cells, but the result shows CD10-, indicating B cell abnormality; light chain deletion indicates B cell abnormality.

[0116] The significant increase in IL-10 and IL-6 indicated B-cell lymphoma. After examination, the patient was transferred to a specialized cancer hospital for further treatment.

[0117] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. Use of an intraocular lymphoma detection reagent in the preparation of an intraocular lymphoma detection product, characterized in that: The steps include: (1) Sample preparation: Taking an intraocular fluid sample, centrifuging it, and removing the supernatant to obtain a concentrated sample; (2) Initial screening: The concentrated sample and the first reagent are mixed evenly, and then incubated in the dark, followed by washing with PBS buffer, centrifugation is continued, the supernatant is removed, and then PBS buffer is added and resuspended to obtain a test sample A; the first reagent includes CD117 antibody, CD56 antibody, CD45 antibody, CD38 antibody, CD34 antibody, CD33 antibody, CD19 antibody, CD13 antibody, CD7 antibody, kappa antibody and lambda antibody; the test sample A is tested by flow cytometry to obtain data; (3) Typing, determining the antibody combination used in the second reagent based on the results of the primary screening in (2); The concentrated sample and the second reagent were mixed evenly, and then incubated in the dark, followed by washing with PBS buffer, centrifugation continued, the supernatant was removed, and then PBS buffer was added and resuspended to obtain the test sample B; Using flow cytometry to test sample B and obtain data; The second reagent includes CD16 antibody, CD22 antibody, CD4 antibody, CD5 antibody, CD23 antibody, CD20 antibody, CD3 antibody, CD138 antibody, CD19 antibody, CD8 antibody, CD45 antibody, CD300e antibody, CD64 antibody, CD123 antibody, CD85K antibody, CD14 antibody, CD36 antibody, CD2 antibody, CD11b antibody, CD303 antibody, CD71 antibody, CD304 antibody, CD10 antibody, MPO antibody, CD79a antibody, FMC7 antibody, IgM antibody, CD103 antibody, CD95 antibody, CD39 antibody, CD11c antibody, CD30 At least twenty of the group consisting of antibodies, CD25 antibodies, CD40 antibodies, CD43 antibodies, BCL-2 antibodies, CD200 antibodies, ki-67 antibodies, CD56 antibodies, CD38 antibodies, Kappa antibodies, Lambda antibodies, CD27 antibodies, CD81 antibodies, CD28 antibodies, TdT antibodies, CD9 antibodies, CD66c antibodies, HLA-DR antibodies, CD307d antibodies, CD1a antibodies, CD57 antibodies, CD45RA antibodies, CD158e1 antibodies, CD158e2 antibodies, CD158b antibodies, CD158i antibodies, CD158f antibodies, CD158a antibodies, and CD158h antibodies.

2. The use of an intraocular lymphoma detection reagent according to claim 1 in the preparation of an intraocular lymphoma detection product, characterized in that: (3) The classification also includes the following steps: Take the third reagent, mix the cytokine microspheres and microsphere buffer, and incubate; Then, the concentrated sample and antibody were added, mixed evenly, incubated, and then PBS buffer was added for washing, centrifuged, the supernatant was removed, and then PBS buffer was added for resuspending to obtain the test sample C; Using flow cytometry to test sample C and obtain data; The third reagent includes a kit for detecting IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α in intraocular fluid.

3. Use of an intraocular lymphoma detection reagent according to claim 1 in the preparation of an intraocular lymphoma detection product, characterized in that: Each antibody in the first reagent is labeled with a fluorescent marker; and each antibody selected in the second reagent is labeled with a fluorescent marker.

4. Use of an intraocular lymphoma detection reagent according to claim 1 in the preparation of an intraocular lymphoma detection product, characterized in that: The intraocular fluid sample is aqueous humor or vitreous humor.

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