Method and kit for detecting lymphocyte proliferation conditions through non-diagnostic purpose

Abstract

The invention relates to the field of cell proliferation detection and a method and a kit for detecting lymphocyte proliferation conditions through a non-diagnostic purpose. The method comprises the following steps: mixing a first to-be-detected blood sample with a lymphocyte polyclonal activator, and performing cell culture, thereby obtaining first blood cells; incubating a fluorescence-labeled lymphocyte surface labeled antibody with the first blood cells, performing red cell lysis, adding microspheres for absolute counting, detecting the number of lymphocytes and the number of the microspheres for absolute counting by adopting flow cytometry, and obtaining the concentration of lymphocytes in a stimulation group according to the number of lymphocytes, the number of the microspheres for absolute counting and the concentration of the microspheres for absolute counting. The proliferation ratio of each subgroup of the lymphocytes can be detected, the absolute value of proliferation of each subgroup of the lymphocytes also can be detected, and the proliferation conditions of the lymphocytes can be accurately reflected; a proliferation experiment is performed by using whole blood, PBMC does not need to be separated, and the requirement on the amount of the blood sample is small; and moreover, the operating steps are simple, the time consumption is low, and the error is small.

Description

technical field [0001] The invention relates to the field of cell proliferation detection, in particular to a method for detecting lymphocyte proliferation for non-diagnostic purposes and a kit thereof. Background technique [0002] Primary immunodeficiency disease (PID) is a disease in which the mutation of related genes leads to changes in the quantity or quality of immune cells or their components, resulting in a significantly increased susceptibility of the body to various pathogens. Among the more than 200 kinds of PIDs that have been discovered so far, more than 200 gene mutations have been identified. The assessment of immune function is of great significance to the diagnosis and prognosis of PID. On the one hand, the evaluation of immune function in children with genetically confirmed PID is helpful to judge the severity of the children's condition, guide clinical medication, and provide reference for clinical treatment and hematopoietic stem cell transplantation; o...

AI Technical Summary

Problems solved by technology

Its disadvantages include: labeling with radionuclides, there is a risk of radioactive contamination; this method can only analyze the proliferation of total lymphocytes, but cannot judge the proliferation of lymphocyte subsets; PBMCs need to be separated, and the blood sample volume is relatively large (usually >4mL), it is more difficult to collect specimens in young patients; this method can only calculate the proportion of lymphocyte proliferation, and cannot obtain the absolute value of cell proliferation

[0009] The principle of the nucleoside analog (BrDU) bromodeoxyuridine incorporation method is similar to that of the radionuclide incorporation method, and its disadvantages include: the sensitivity of the method is not high; the method can only analyze the total lymphocytes The proliferation of lymphocyte subsets cannot be judged; PBMCs need to be separated, and the blood sample volume is relatively large (usually >4mL), and it is more difficult to collect samples in young patients; this method can only calculate the proliferation ratio of lymphocytes, and cannot get the absolute value of cell proliferation

Its shortcomings include: this method is an indirect method for assessing the proliferation of lymphocytes, by calculating the ratio of living cells, rather than directly detecting the number of dividing cells; this method can only analyze the proliferation of total lymphocytes, and cannot judge Proliferation of lymphocyte subsets; PBMC need to be separated, and the blood sample volume is relatively large (usually >4mL), and it is more difficult to collect samples in young patients; this method can only calculate the proportion of lymphocyte proliferation, and cannot obtain the absolute value of cell proliferation value

Its disadvantages include: separation of PBMCs is required, the volume of blood samples is relatively large (usually >4mL), and it is more difficult to collect samples in young patients; after the extraction of PBMCs, it is usually followed by multiple washings, which may be activated, damaged, or Cells are selectively lost; this method can only calculate the proportion of lymphocyte proliferation, and cannot obtain the absolute value of cell proliferation; the operation steps are complicated, and the error is large, and this method is based on the initial PBMC quantity in the stimulation group and the non-stimulation group ( Rather than the number of lymphocytes) are the same, and subsequent repeated washing and pipetting may introduce more errors; CFSE staining requires uniformity, and CFSE has certain toxicity at high concentrations

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