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A kind of isotonic hemolysin and its preparation method, biological sample processing method and leukocyte membrane antigen detection method

A technology of biological samples and hemolysin, which is applied in the field of immunological cell analysis, can solve problems such as inability to achieve hemolysin effect, differences in white blood cell morphology, and differences in granulocyte grouping

Active Publication Date: 2022-02-18
GENERAL HOSPITAL OF NUCLEAR IND
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Problems solved by technology

[0004] The formula of hemolysin has a certain relationship with the instrument. Different instruments use different hemolysin. The same hemolysin shows different effects on different instruments. The main reason is that the leukocyte morphology after hemolysin lysis is different. The optical path design will lead to differences in lymphoid, monocyte and granulocyte grouping
At present, many hemolysins on the market are only suitable for a certain type of flow cytometer, and cannot achieve the role of a hemolysin universal multi-type flow cytometer

Method used

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  • A kind of isotonic hemolysin and its preparation method, biological sample processing method and leukocyte membrane antigen detection method
  • A kind of isotonic hemolysin and its preparation method, biological sample processing method and leukocyte membrane antigen detection method
  • A kind of isotonic hemolysin and its preparation method, biological sample processing method and leukocyte membrane antigen detection method

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preparation example Construction

[0030] On the basis of the above-mentioned isotonic hemolysin, the present invention also discloses a preparation method of isotonic hemolysin, comprising the following steps:

[0031] S1. Take sodium nitrite, sodium chloride, calcium chloride and magnesium chloride according to the above content and add them into the water medium to fully dissolve to obtain a mixed solution.

[0032]S2. Add formaldehyde, glycerol, and isobutanol solution to the mixed buffer solution obtained in step S1, add water medium to 1 liter, adjust the pH value to 7.4-7.6 with sodium bicarbonate, and then filter to obtain hemolysin.

[0033] The method of treating the biological sample with the above-mentioned isotonic hemolysin is as follows: add the hemolysin to the biological sample at room temperature, mix and treat for 10-15 minutes; the biological sample is anticoagulant venous whole blood; The volume ratio of the hemolysin to the biological sample is 1:40-3:40.

[0034] Meanwhile, the method fo...

Embodiment

[0049]

[0050] Accuracy experiment:

[0051] In order to measure the inaccuracy of the results and evaluate the size of the system error, the following experiments were carried out: 15 anticoagulated peripheral blood samples from healthy and sub-healthy people were selected, each sample was measured twice with two hemolysins, and the experimental results were recorded , for linear regression and correlation analysis. Table 1 Correlation analysis of the results of T lymphocyte subsets detected by self-made hemolysin and OptiLyse C hemolysin (%)

[0052]

[0053]

[0054] Precision experiment:

[0055] In order to evaluate the size of the random error in the measurement results, the following experiments were carried out: 1. Repeated test within the batch: use Immuno-Trol cells quality control blood to add samples continuously for 10 times in the same time period with two kinds of hemolysin; 2. Batch Interval repeated test: Immuno-Trol cells quality control blood was...

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Abstract

This isotonic hemolysin includes oxidation dissolving agent, cosolvent, cell solubilizing agent, cell fixative, inorganic salt and water quality, oxidation dissolving agent is sodium nitrite, and content is 2-20g / L; One or more of ethylene glycol and propylene glycol, and the content is 0.3-3mmol / L; the cell solubilizer is one or more of isobutanol, methanol, and ethanol, and the content is 0.1-1mol / L; Fixing agent is one or more of paraformaldehyde, formaldehyde, ethanol, acetone, and content is 10-40g / L; Inorganic salt is sodium chloride, magnesium chloride and calcium chloride, and the content of described sodium chloride is 1-10g / L, the content of magnesium chloride is 1-100mmol / L, and the content of calcium chloride is 1-100mmol / L. At the same time, a method for preparing the hemolysin, a method for treating biological samples with the hemolysin, and a method for detecting leukocyte membrane antigens by flow cytometry were proposed. Biological activity, and can completely lyse red blood cells, with accurate results and stable performance.

Description

technical field [0001] The invention relates to the field of immunological cell analysis, in particular to an isotonic hemolysin and a preparation method thereof, a method for processing biological samples and a method for detecting white blood cell membrane antigens. Background technique [0002] Blood cell analysis is of great value for scientific research, disease prevention, diagnosis and treatment, from the initial study of cell shape, size, quantity to the most recent antigenic composition on the cell surface. Traditional cellular immunity and non-specific immune function detection techniques cannot perform multi-parameter and high-sensitivity analysis on the size, shape, plasma membrane, and internal structure of target cells from the single-cell level. Flow cytometry can quickly measure, sort, Store and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquid, and can sort out specified cell subpopulat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/06
CPCG01N33/533G01N15/1404G01N15/1434
Inventor 俞秋兴
Owner GENERAL HOSPITAL OF NUCLEAR IND
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