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Immunofluorescence detection method for PD-L1 expression on peripheral blood circulating tumor cells of hepatocellular carcinoma patient

A technology of peripheral blood circulation and PD-L1, which is applied in the field of molecular biology, can solve the problem that obtaining tissue samples cannot evaluate the real-time dynamic state of PD-L1 in patients, and achieve the effects of avoiding edge effects, good stability, and reducing cell loss

Pending Publication Date: 2020-10-02
SHANDONG RES INST OF TUMOUR PREVENTION TREATMENT
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Problems solved by technology

[0007] Aiming at the shortcomings in the prior art that patients with advanced or recurrent hepatocellular carcinoma cannot obtain tissue samples in real time or repeated punctures, and thus cannot evaluate the real-time dynamic state of PD-L1 in patients, the present invention provides a method for detecting PD in circulating tumor cells in peripheral blood of patients with hepatocellular carcinoma. -L1 expression kit and detection method: Use a membrane filtration device to separate and obtain circulating tumor cells (CTCs) in the peripheral blood of patients with advanced hepatocellular carcinoma, and further use immunofluorescence technology to detect the expression of PD-L1 in CTCs

Method used

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  • Immunofluorescence detection method for PD-L1 expression on peripheral blood circulating tumor cells of hepatocellular carcinoma patient
  • Immunofluorescence detection method for PD-L1 expression on peripheral blood circulating tumor cells of hepatocellular carcinoma patient

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Embodiment 1

[0044] 1. Rinsing of the filter of circulating tumor cell separator (YZYMED-A10)

[0045] a) Open the upper and lower plugs of the filter and place the filter on the rinse stand;

[0046] b) Add about 2mL of 75% medical alcohol into the filter with a Pasteur tube and wait for it to naturally filter out from the bottom of the filter;

[0047] c) Add 8 mL of normal saline to the filter for rinsing, remove the residual alcohol in the filter, and naturally filter out the normal saline;

[0048] d) Repeat step 3 twice, leave an appropriate amount of saline to ensure that the filter membrane is wet, and install the lower plug of the filter;

[0049] Note: If the alcohol does not filter out naturally or the filter out is too slow during the filter rinsing process, please discard the filter immediately.

[0050] 2. Using immunofluorescence to detect the expression of PD-L1 in peripheral blood circulating tumor cells of patients with hepatocellular carcinoma:

[0051] (1) Peripheral...

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Abstract

The invention discloses an immunofluorescence detection method for PD-L1 expression on peripheral blood circulating tumor cells of a hepatocellular carcinoma patient. A kit comprises 100 [mu]l of a primary antibody suspension composed of 0.4% of PFA, 0.3% of TritonX-100, 10% of goat serum, anti-human PD-L1, and rat anti-CD45, 100 [mu]l of a secondary antibody suspension composed of a fluorescentlylabeled goat anti rabbit, and a fluorescently labeled goat anti rat, and a DAPI mounting medium. The detection method comprises the following steps: peripheral blood collection, peripheral blood sample pretreatment, sample separation, methanol fixation, film pasting, stroke circle grouping, cell fixation, cell penetration, sealing, primary antibody incubation, secondary antibody incubation and nuclear staining, sealing and detection. According to the detection method provided by the invention, the PD-L1 expression condition of a patient with advanced or recurrent hepatocellular carcinoma canbe detected without acquiring a tissue specimen through needle biopsy, and real-time dynamic detection can be realized by utilizing a minimally invasive technology.

Description

technical field [0001] The invention provides a kit and detection method for detecting PD-L1 expression in peripheral blood circulating tumor cells of patients with hepatocellular carcinoma, and provides real-time and dynamic clinical guidance for patients with advanced hepatocellular carcinoma to apply PD-1 inhibitor immunotherapy, which belongs to molecular biology technology field. Background technique [0002] Liver cancer is one of the common malignant tumors in my country, with a high mortality rate. It ranks third in the death order of malignant tumors after stomach and esophagus; it ranks second in rural areas in some areas, second only to gastric cancer. About 110,000 people die from liver cancer every year in my country, accounting for 45% of the world's liver cancer deaths. The average age of liver cancer is 44 years old. The pathological types of liver cancer are hepatocellular carcinoma, cholangiocarcinoma, and mixed liver cancer, among which hepatocellular car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/574
CPCG01N33/582G01N33/57438G01N33/57492G01N2333/70532
Inventor 崔凯李胜李浩马莹戚元刚
Owner SHANDONG RES INST OF TUMOUR PREVENTION TREATMENT
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