CN112986565A - Kit for rapidly detecting peripheral T cell lymphoma and use method thereof - Google Patents
Kit for rapidly detecting peripheral T cell lymphoma and use method thereof Download PDFInfo
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- CN112986565A CN112986565A CN202110300174.XA CN202110300174A CN112986565A CN 112986565 A CN112986565 A CN 112986565A CN 202110300174 A CN202110300174 A CN 202110300174A CN 112986565 A CN112986565 A CN 112986565A
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Abstract
The invention belongs to the field of immunological detection, relates to a kit, and particularly relates to a kit for rapidly detecting peripheral T cell lymphoma and a using method thereof. The kit comprises a reagent I and a reagent II, wherein the reagent I comprises two groups of monoclonal antibody mixtures, and the first group of monoclonal antibody mixtures comprises the following components: TRBC1, CD2, CD3, CD5, CD7, CD16, CD56, CD57, and CD 45; the second group of monoclonal antibody mixture comprises the following components: TRBC1, TCR- γ δ, CD3, CD8, CD4, CD7 and CD 45; and the reagent II is hemolysin. The kit provided by the invention realizes rapid and accurate detection of Peripheral T Cell Lymphoma (PTCL), is simple and convenient, greatly reduces the requirement on the sample size and reduces the burden of patients.
Description
Technical Field
The invention belongs to the field of immunological detection, relates to a kit, and particularly relates to a kit for rapidly detecting peripheral T cell lymphoma and a using method thereof.
Background
At present, immunophenotyping diagnosis of T cell lymphoma depends on loss or abnormality of T cell surface antigen, and T cell lymphoma is various in types, complex in classification and difficult in pathological diagnosis. These changes are often very subtle, and the diagnosis of certain T-cell lymphomas is poorly characterized by HE and immunohistochemical staining alone; while flow cytometry can detect deletions, abnormal increases or decreases in T cell antigen expression to aid in the diagnosis of T cell lymphomas, certain T cell lymphomas do not have significant phenotypic abnormalities or can have phenotypic abnormalities in reactive T cells activated upon infection and in autoimmune diseases, thus further testing for T cell clonality is desirable.
The existing detection means for diagnosing the clonality of the T cells comprise TCR gene rearrangement detection and flow TCR-V beta detection. The PCR amplification method is commonly used for carrying out TCR gene rearrangement analysis clinically, the accuracy of the TCR gene rearrangement is influenced by the limited diversity of the TCR gene rearrangement, high-level background noise amplification, lack of clone quantification or immunophenotyping characteristics and the like, and the domestic TCR rearrangement detection technology with unified regulation is not available, so the detection time is too long, and the cost is high.
The traditional flow type TCR-V beta detection needs 8-tube detection, the required sample size is large, most patients find suspicious T cell subsets when FCM primary screening is finished, the residual sample size is not enough to be added into 8 tubes, the clinical sample extraction needs to be informed, if the sample is bone marrow and the patients do not cooperate with repeated sample extraction, only peripheral blood is used for replacing the bone marrow for detection, but the difference between the peripheral blood and the bone marrow can influence the result; and in the detection method, 24 antibodies and 8 tubes of samples are used for detection, so that the antibody cost is high, the operation complexity is high, the analysis time is long, and the diagnosis and treatment of patients with urgent disease conditions are not facilitated in clinical practice.
Disclosure of Invention
Aiming at the technical problems, the invention provides a kit for rapidly detecting peripheral T cell lymphoma and a using method thereof, and solves the technical problems of large sample size, complex steps and low detection efficiency of the conventional kit.
The technical scheme of the invention is realized as follows:
a kit for rapidly detecting peripheral T cell lymphoma comprises a reagent I and a reagent II, wherein the reagent I comprises two groups of monoclonal antibody mixtures, and the first group of monoclonal antibody mixtures comprises the following components: TRBC1, CD2, CD3, CD5, CD7, CD16, CD56, CD57, and CD 45; the second group of monoclonal antibody mixture comprises the following components: TRBC1, TCR- γ δ, CD3, CD8, CD4, CD7 and CD 45; and the reagent II is hemolysin.
And the reagent II is hemolysin and comprises ammonium chloride, potassium bicarbonate, ethylene diamine tetraacetic acid and ultrapure water.
The specific contents of the first group of monoclonal antibody mixture are as follows: 5 μ L TRBC1, 5 μ L CD2, 5 μ L CD3, 5 μ L CD5, 5 μ L CD7, 5 μ L CD16, 5 μ L CD56, 5 μ L CD57, and 5 μ L CD 45; the second group of monoclonal antibody mixture comprises the following specific contents: 5 μ L TRBC1, 5 μ L TCR- γ δ, 5 μ L CD3, 5 μ L CD8, 5 μ L CD4, 5 μ L CD7 and 5 μ L CD 45.
The hemolysin comprises the following components in percentage by mass: 0.821% of ammonium chloride, 0.0991% of potassium bicarbonate, 0.00367% of ethylenediamine tetraacetic acid and the balance of ultrapure water.
The use method of the kit comprises the following steps:
(1) adding a sample to be detected into a detection container, then adding a certain volume of reagent II, uniformly oscillating, incubating for 10-20min at room temperature in a dark place, and then centrifuging to remove cell debris and non-cell components;
(2) adding sheath fluid into the sample to be detected processed in the step (1), adding a certain volume of reagent I after cell counting is achieved, incubating for 15min at room temperature, adding 2mL of sheath fluid, centrifuging again, adding 500 mu L of sheath fluid for heavy suspension, and placing in a flow cytometer for detection;
(3) and (3) screening whether the T cell subset and the reactive lymphocyte TCR beta of the sample to be detected present monomorphic expression or not according to the detection result of the step (2), thereby realizing rapid diagnosis.
The volume ratio of the sample to be detected to the reagent II in the step (1) is 1: 20; the speed of centrifugation was 1700 r/min.
The sheath liquid in the step (2) is BD 342003; cell counts were normalized to include per tubeHas (0.8-1.2) × 106And (4) cells.
The adding amount of the reagent I in the step (2) is 20 mu L.
If the T cell subgroup and the reactive lymphocyte TCR beta are expressed in a monomorphic way in the step (3), the T cell subgroup and the reactive lymphocyte TCR beta are peripheral T cell lymphoma cells; if T cell subset, reactive lymphocyte TCR beta is expressed in polymorphism, then it is normal cell.
The invention has the following beneficial effects:
1. the kit is constructed by combining TRBC1 antibodies, and the expression conditions of TRBC1 in T cell subsets and reactive lymphocytes are measured based on the monomorphic expression of a T cell lymphoma (PTCL) receptor beta chain constant region, so that the Peripheral T Cell Lymphoma (PTCL) can be rapidly distinguished and detected; the application reduces the types of monoclonal antibodies of the kit, only uses 9 antibodies, realizes the accurate detection of the clonality of T cells of mature T cell lymphoma, and enhances the specific recognition capability of the T cells, thereby realizing the rapid and accurate detection of Peripheral T Cell Lymphoma (PTCL); the kit of the application can greatly reduce the number of required samples and reduce the double damage to the mind and body of a patient.
2. The kit of the application completes the inspection through conventional operation, has no complicated steps, shortens the detection period, reduces the detection cost, and reduces the technical requirements on medical detection personnel through conventional operation, thereby carrying out rapid detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows that the abnormal mature T lymphocyte TRBC1 is expressed in a haplotype.
FIG. 2 shows the polymorphism expression of TRBC1 in normal mature T lymphocytes.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
A kit for rapidly detecting peripheral T cell lymphoma comprises a reagent I and a reagent II, wherein the reagent I comprises two groups of monoclonal antibody mixtures, and the first group of monoclonal antibody mixtures comprises the following components: TRBC1, CD2, CD3, CD5, CD7, CD16, CD56, CD57, and CD 45; the second group of monoclonal antibody mixture comprises the following components: TRBC1, TCR- γ δ, CD3, CD8, CD4, CD7 and CD 45; and the reagent II is hemolysin.
Further, the reagent II is hemolysin, and comprises ammonium chloride, potassium bicarbonate, ethylene diamine tetraacetic acid and ultrapure water.
Further, the specific contents of the first group of monoclonal antibody mixture are as follows: 5 μ L TRBC1, 5 μ L CD2, 5 μ L CD3, 5 μ L CD5, 5 μ L CD7, 5 μ L CD16, 5 μ L CD56, 5 μ L CD57, and 5 μ L CD 45; the second group of monoclonal antibody mixture comprises the following specific contents: 5 μ L of RBC1, 5 μ L of TCR-. gamma.δ, 5 μ L of CD3, 5 μ L of CD8, 5 μ L of CD4, 5 μ L of CD7 and 5 μ L of CD 45.
Further, the hemolysin comprises the following components in percentage by mass: 0.821% of ammonium chloride, 0.0991% of potassium bicarbonate, 0.00367% of ethylenediamine tetraacetic acid and the balance of ultrapure water.
The use method of the kit comprises the following steps:
(1) adding a sample to be detected into a detection container, then adding a certain volume of reagent II, uniformly oscillating, incubating for 10-20min at room temperature in a dark place, and then centrifuging to remove cell debris and non-cell components;
(2) adding sheath fluid into the sample to be detected processed in the step (1), adding a certain volume of reagent I after cell counting reaches the standard, incubating for 15min at room temperature, adding 2mL of sheath fluid, centrifuging again, adding 500 mu L of sheath fluid for heavy suspension, and placing in a flow cytometer for detection;
(3) and (3) screening whether the T cell subset and the reactive lymphocyte TCR beta of the sample to be detected present monomorphic expression or not according to the detection result of the step (2), thereby realizing rapid diagnosis.
Further, the volume ratio of the sample to be detected to the reagent II in the step (1) is 1: 20; the speed of centrifugation was 1700 r/min.
Further, the sheath fluid in the step (2) is BD 342003; cell counts were normalized to contain (0.8-1.2). times.10 cells per tube6And (4) cells.
Further, the amount of the reagent I added in the step (2) is 20. mu.L.
Further, in the step (3), if the T cell subset, namely the reactive lymphocyte TCR β, is expressed in a haplotype, the T cell subset is a peripheral T cell lymphoma cell; if T cell subset, reactive lymphocyte TCR beta is expressed in polymorphism, then it is normal cell.
Example 1
An example of a clinical sample for which a clear clinical diagnosis is shown in FIG. 1 was tested using the kit of the present application. This example employs a flow cytometer. The operation steps of the test are as follows:
adding hemolysin with the volume equivalent of about 1:20 into 100 mu L of sample, incubating for 10 minutes in the dark at room temperature, removing cell debris or non-cell components, repeating the operation to prepare two samples, adding a small amount of sheath solution to dilute, adding 20 mu L of the mixture of the two groups of monoclonal antibodies into the two samples one by one at room temperature, incubating for 15 minutes in the dark, adding 500 mu L of PBS solution at room temperature, incubating for 5 minutes in the dark at room temperature, centrifuging, diluting with a proper amount of sheath solution, and detecting on a machine.
The experiment yielded assay data as shown in FIG. 1. The T cell TCR beta of the tumor is expressed in a monomorph, and TRBC1 can identify the clonality of the T cell subgroup with high sensitivity, so that the T cell of the tumor can be distinguished from the T cell of the non-tumor.
Example 2
The kit is used for detecting a normal clinical sample by adopting a flow cytometer, and the specific operation steps of the flow cytometer are as follows: respectively taking 100 mu L of sample, adding hemolysin with the volume equivalent of about 1:20, incubating for 15min in the dark at room temperature, centrifuging for two times to remove cell debris and non-cell components, adding a small amount of sheath fluid, adding two groups of monoclonal antibody mixed solution into 20 mu L one by one, incubating for 15min at room temperature, adding 2mL of sheath fluid, centrifuging again, adding 500 mu L of sheath fluid, and performing detection for two times.
Final test results as shown in fig. 2, the TRBC1 antibody detection results exhibited partial expression in normal samples.
The result shows that the TRBC1 antibody kit used in the patent can realize sensitive diagnosis of PTCL patients, and can simply and accurately screen T cell subsets and whether the TCR beta of reactive lymphocytes presents monomorphic expression, thereby realizing rapid diagnosis and providing basis for subsequent treatment.
Example 3
25 samples of mature T cell lymphoma invading bone marrow were tested simultaneously using the kit of the present application.
In this example, a flow cytometer is used, and the operation steps of each test are as follows:
and (3) taking 20 mu L of monoclonal antibody mixture and 100 mu L of sample, incubating for 15 minutes at room temperature in a dark place, adding hemolysin with the volume equivalent of about 1:20, incubating for 10 minutes at room temperature in a dark place, adding 500 mu L of PBS solution, incubating for 5 minutes at room temperature in a dark place, and detecting on a machine. Repeating the steps to obtain 25 groups of detection data;
as shown in table 2:
TABLE 2 25 sets of data obtained in example 3
It is generally believed that less than 15% or greater than 85% of cells expressing TRBC1 express the TCR β as unitype. The final test results of 25 samples show that the TRBC1 expression of the test results of the samples with the T-cell lymphoma is in monomorphic expression, and as shown in samples 23-25, the content of the tested TRBC1 is more than 90%, so that rapid and accurate diagnosis is realized. The data result shows that the TRBC1 can identify the clonality of the T cell subgroup with high sensitivity, and the kit has universal applicability and accurate detection on samples.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. The kit for rapidly detecting the peripheral T cell lymphoma is characterized by comprising a reagent I and a reagent II, wherein the reagent I comprises two groups of monoclonal antibody mixtures, and the first group of monoclonal antibody mixtures comprise the following components: TRBC1, CD2, CD3, CD5, CD7, CD16, CD56, CD57, and CD 45; the second group of monoclonal antibody mixture comprises the following components: TRBC1, TCR- γ δ, CD3, CD8, CD4, CD7 and CD 45; and the reagent II is hemolysin.
2. The kit of claim 1, wherein: and the reagent II is hemolysin and comprises ammonium chloride, potassium bicarbonate, ethylene diamine tetraacetic acid and ultrapure water.
3. The kit according to claim 2, wherein the first mixture of mabs is present in an amount specified by: 5 μ L TRBC1, 5 μ L CD2, 5 μ L CD3, 5 μ L CD5, 5 μ L CD7, 5 μ L CD16, 5 μ L CD56, 5 μ L CD57, and 5 μ L CD 45; the second group of monoclonal antibody mixture comprises the following specific contents: 5 μ L TRBC1, 5 μ L TCR- γ δ, 5 μ L CD3, 5 μ L CD8, 5 μ L CD4, 5 μ L CD7 and 5 μ L CD 45.
4. The kit according to claim 2, wherein the hemolysin comprises the following components in percentage by mass: 0.821% of ammonium chloride, 0.0991% of potassium bicarbonate, 0.00367% of ethylenediamine tetraacetic acid and the balance of ultrapure water.
5. The method of using the kit of any one of claims 1 to 4, characterized in that the steps are as follows:
(1) adding a sample to be detected into a detection container, then adding a certain volume of reagent II, uniformly oscillating, incubating for 10-20min at room temperature in a dark place, and then centrifuging to remove cell debris and non-cell components;
(2) adding sheath fluid into the sample to be detected processed in the step (1), adding a certain volume of reagent I after cell counting reaches the standard, incubating for 15min at room temperature, adding 2mL of sheath fluid, centrifuging again, adding 500 mu L of sheath fluid for heavy suspension, and placing in a flow cytometer for detection;
(3) and (3) screening whether the T cell subset and the reactive lymphocyte TCR beta of the sample to be detected present monomorphic expression or not according to the detection result of the step (2), thereby realizing rapid diagnosis.
6. Use according to claim 5, characterized in that: the volume ratio of the sample to be detected to the reagent II in the step (1) is 1: 20; the speed of centrifugation was 1700 r/min.
7. Use according to claim 5, characterized in that: the sheath liquid in the step (2) is BD 342003; cell counts were normalized to contain (0.8-1.2). times.10 cells per tube6And (4) cells.
8. Use according to claim 7, characterized in that: the adding amount of the reagent I in the step (2) is 20 mu L.
9. Use according to claim 5, characterized in that: if the T cell subgroup and the reactive lymphocyte TCR beta are expressed in a monomorphic way in the step (3), the T cell subgroup and the reactive lymphocyte TCR beta are peripheral T cell lymphoma cells; if T cell subset, reactive lymphocyte TCR beta is expressed in polymorphism, then it is normal cell.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113777327A (en) * | 2021-09-13 | 2021-12-10 | 北京大学人民医院 | Antibody composition for leukemia/lymphoma immunophenotyping primary screening and application thereof |
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2021
- 2021-03-22 CN CN202110300174.XA patent/CN112986565A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| HOLLY BERG, ET AL.,: "Flow cytometric evaluation of TRBC1 expression in tissue specimens and body fluids is a novel and specific method for assessment of T-cell clonality and diagnosis of T-cell neoplasms", CYTOMETRY PART B: CLINICAL CYTOMETRY * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113777327A (en) * | 2021-09-13 | 2021-12-10 | 北京大学人民医院 | Antibody composition for leukemia/lymphoma immunophenotyping primary screening and application thereof |
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