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Detection method of decidual lymphocyte subset, kit and application of kit

A technology of lymphocytes and detection methods, applied in the field of detection of lymphocyte subtypes, can solve the problems of large amount of antibodies, large demand, long operation steps and labor hours, etc., and achieve the effect of saving antibodies and simple operation.

Inactive Publication Date: 2017-09-08
BEIJING OBSTETRICS & GYNECOLOGY HOSPITAL CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method consumes a large amount of antibodies, and the operation steps and man-hours are many. Multiple antibodies are used repeatedly to conduct comprehensive antigen analysis on patients, and the cost is relatively high.
At the same time, there is a large demand for decidual lymphocyte samples to be tested, and a sufficient amount of decidual tissue needs to be collected to complete the test. However, the patient's decidual tissue needs to be obtained through surgery in a sterile environment, and the number of isolated lymphocytes is limited.
These problems bring a lot of inconvenience to the detection of decidual lymphocyte subtypes in patients with RM

Method used

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  • Detection method of decidual lymphocyte subset, kit and application of kit
  • Detection method of decidual lymphocyte subset, kit and application of kit
  • Detection method of decidual lymphocyte subset, kit and application of kit

Examples

Experimental program
Comparison scheme
Effect test

no. 1 example

[0043] 1. Preparation of decidual lymphocyte samples

[0044] 1. Isolation of decidua mononuclear cells (dMC)

[0045] 1.1. The size of the tissue block is about 2×1×1cm, cut it into 1×1×1mm pieces with a blade, put it into a tissue grinder, add 1mL of culture medium, turn the pestle, grind until homogenized, Rinse the grinder twice with 5mL of culture solution, pass through a cell strainer, filter, put into a 10mL centrifuge tube, and centrifuge at 450g for 5min;

[0046] 1.2. Resuspend the cell mass with 2mL of culture medium, spread on 2mL of density gradient separation medium, and centrifuge at 2000rpm for 15min without interruption;

[0047] 1.3. Carefully draw the cells at the interface into a 1mL centrifuge tube, centrifuge at 2000rpm for 10min, remove the supernatant, resuspend the cells in 0.5mL culture medium, and flick the bottom of the centrifuge tube fully.

[0048] Among them, when the amount of cells is insufficient, the remaining tissue after grinding can be ...

no. 2 example

[0109] Using the method described in the first embodiment, decidua lymphocyte subtypes were detected on 31 samples collected from 10 cases of URM admitted to Beijing Obstetrics and Gynecology Hospital Affiliated to Capital Medical University from August 2016 to February 2017 Patients, with an average age of 31.6±2.5 years, and 21 healthy women who underwent active abortion at the same time, with an average age of 29.6±2.8 years. The cell proportions of decidual lymphocyte subtypes detected by different gating strategies in healthy women and URM patients are shown in Table 2 and Table 3 below, respectively. The corresponding cell number can be reflected by the cell ratio.

[0110] Table 2: Cell Proportions of Decidua Lymphocyte Subtypes in Healthy Women

[0111]

[0112] Table 3: Proportions of cells of decidua lymphocyte subtypes in URM patients

[0113]

[0114] The data in Table 2 and Table 3 were analyzed by 2 × 2 factorial design analysis of variance. The results s...

no. 3 example

[0119] The third embodiment: the application of the kit for detecting subtypes of decidual lymphocytes in the immunological detection of recurrent spontaneous abortion.

[0120] method:

[0121] a. Use the kit for detecting decidual lymphocyte subtypes described in the first embodiment, and detect the decidual lymphocyte subtypes of healthy people and patients with repeated spontaneous abortion according to the method described in the first embodiment, to obtain Test results;

[0122] b. Comparing the cell ratios of Th1 cell subgroups, Tc1 cell subgroups, dNK1 cell subgroups and / or NKT1-like cell subgroups in the decidual lymphocyte subtypes of the healthy population and the recurrent spontaneous abortion patients in the test results ;as well as

[0123] c. If the cell ratio of the Th1 cell subgroup, Tc1 cell subgroup, dNK1 cell subgroup and / or NKT1-like cell subgroup in the patient with recurrent spontaneous abortion is higher than the average cell ratio of the cell subgrou...

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Abstract

The invention provides a detection method of a decidual lymphocyte subset. The detection method comprises the following steps: carrying out fluorescent staining on the same decidual lymphocyte subset sample by virtue of a fluorescently-labeled anti-CD3 antibody, a fluorescently-labeled anti-CD4 antibody, a fluorescently-labeled anti-CD8 antibody, a fluorescently-labeled anti-CD56 antibody, a fluorescently-labeled anti-CD16 antibody, a fluorescently-labeled anti-IFN-gamma antibody, a fluorescently-labeled anti-IL-4 antibody and Fixable Viability Dye, so as to obtain the stained sample; detecting the stained sample by virtue of a flow cytometry, so as to obtain detection data; and analyzing the detection data. The detection method has the beneficial effects that fewer decidual lymphocyte samples are required, antibodies are saved, and the operation is simple. Besides, the invention further provides a kit utilizing the detection method and application of the kit in the immunological detection of the recurrent spontaneous abortion.

Description

technical field [0001] The present invention relates to a detection method of lymphocyte subtype, in particular to a detection method of decidua lymphocyte subtype by flow cytometry, a kit using the method and the kit in immunology of repeated spontaneous abortion. application in detection. Background technique [0002] Spontaneous abortion is the most common complication of early pregnancy. Recurrent miscarriage (recurrent miscarriage, RM) refers to two or more spontaneous abortions, and the incidence rate accounts for 1%-2% of women of childbearing age. At present, the etiology of RM mainly includes: chromosomal abnormalities in parents or offspring, abnormal uterine anatomy, endocrine disorders, infection, and maternal autoimmunity. However, there are still many unknown recurrent miscarriage (URM) with unknown causes. There is immune tolerance between mother and fetus, and the destruction of this immune tolerance is considered to be one of the causes of RM. Immunothera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N15/14
CPCG01N33/533G01N15/1434G01N2015/1438
Inventor 董鹏李坚
Owner BEIJING OBSTETRICS & GYNECOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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