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Sperm quality evaluation method based on flow cytometry

A technology of flow cytometry and sperm quality, applied in measurement devices, individual particle analysis, scientific instruments, etc., can solve the problems of general evaluation and analysis experimental parameters, lack of scientific judgment of sperm cells, limited number of statistical analysis, etc. Multi-parameter joint detection, reducing the intensity of detection work, and increasing the number of statistics

Inactive Publication Date: 2012-08-22
广州华银医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If you want to detect multiple traits of cells, you need to use multiple fluorescent markers, but if the wavelengths of multiple fluorescent markers are relatively close, the flow cytometer cannot distinguish the fluorescence intensities of different wavelengths, resulting in mutual interference of fluorescence scattering signals. interference overlap
[0004] Therefore, the shortcomings of the prior art are as follows: (1) the number of statistical analyzes is limited, and the authenticity of the analysis results is not enough; (2) human factors have a greater impact on the results, and the accuracy of the analysis results is not enough; (3) due to The statistical quantity of the analysis is too small. In order to ensure the accuracy and repeatability of the results, the requirements for the experimental operation before the detection are very high, and the work intensity is relatively high; (4) The experimental parameters for the evaluation and analysis are too general, and most of them are sperm cells. In the expression, there is a lack of scientific judgment on the intrinsic physicochemical properties of sperm cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The sperm quality assessment method based on flow cytometry comprises the following steps:

[0057] 1) Take a freshly liquefied human semen sample, wash it with BSA-PBS buffer, and adjust the number of sperm cells to 10 in the reaction test tube 6 , resuspended into 200 μL cell suspension;

[0058] 2) Add nucleic acid fluorescent dyes Syto16 and 7-AAD at a final concentration of 2 mM to the above-mentioned reaction tubes in sequence, incubate at 37°C for 3 min, and wash with PBS buffer;

[0059] 3) Add APC-labeled mouse anti-human CD46 antibody at a final concentration of 1 mM to the above reaction tube, incubate at 37°C for 30 min, and wash with PBS buffer;

[0060] 4) Add 10 μL of fluorescent microspheres with a diameter of 10 μm to the above reaction test tube, resuspend to 200ul with PBS buffer, set a gate on the flow cytometer with FSC / SSC, perform detection readings, and calculate the final detection results data.

Embodiment 2

[0062] The sperm quality assessment method based on flow cytometry comprises the following steps:

[0063] 1) Take a freshly liquefied human semen sample, wash it with BSA-PBS buffer, and adjust the number of sperm cells in the reaction tube to 3×10 6 , resuspended into 200 μL cell suspension;

[0064] 2) Add nucleic acid fluorescent dyes Syto16 and 7-AAD at a final concentration of 5 mM to the above-mentioned reaction tubes sequentially, incubate at 37°C for 2 min, and wash with PBS buffer;

[0065] 3) Add APC-labeled mouse anti-human CD46 antibody at a final concentration of 3 mM to the above reaction tube, incubate at 37°C for 30 min, and wash with PBS buffer;

[0066] 4) Add 10 μL of fluorescent microspheres with a diameter of 10 μm to the above reaction test tube, resuspend to 200ul with PBS buffer, set a gate on the flow cytometer with FSC / SSC, perform detection readings, and calculate the final detection results data.

Embodiment 3

[0068] The sperm quality assessment method based on flow cytometry comprises the following steps:

[0069] 1) Take a freshly liquefied human semen sample, wash it with BSA-PBS buffer, and adjust the number of sperm cells in the reaction tube to 5×10 6 , resuspended into 200 μL cell suspension;

[0070] 2) Add nucleic acid fluorescent dyes Syto16 and 7-AAD at a final concentration of 3 mM to the above-mentioned reaction tubes in sequence, incubate at 37°C for 5 min, and wash with PBS buffer;

[0071] 3) Add APC-labeled mouse anti-human CD46 antibody at a final concentration of 2mM to the above reaction tube, incubate at 37°C for 25min, and wash with PBS buffer;

[0072] 4) Add 10 μL of fluorescent microspheres with a diameter of 10 μm to the above reaction test tube, resuspend to 200ul with PBS buffer, set a gate with FSC / SSC on the flow cytometer, perform detection readings, and calculate the final detection results data.

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Abstract

The invention discloses a sperm quality evaluation method based on flow cytometry, which comprises the following steps of: firstly, commonly dyeing a semipermeable membrane nucleic acid fluorescent dye, a non-semipermeable membrane fluorescent nucleotide dye and a sample to be detected; and then, incubating a fluorescent marker monoclonal antibody and the dyed sample to be detected and carrying out flow cytometric detection to obtain the final detection results,. The fluorescent marker monoclonal antibody can be reacted with a sperm acrosome membrane. The laser excitation wavelength differences between the fluorescent marker monoclonal antibody and the semipermeable membrane fluorescent nucleotide dye and between the fluorescent marker monoclonal antibody and the non-semipermeable membrane fluorescent nucleotide dye are 100-200 nm, and the fluorescent scattering wavelength differences are 75-150 nm. The sperm quality evaluation method has the beneficial effects through one-time detection, a plurality of parameter results can be simultaneously obtained, so that the apoptosis rate, the plasma membrane integrity rate and the survival rate of sperm are obtained, and the detection labor strength is reduced; and the demands on a large batch of sample detection in the clinical can be satisfied.

Description

technical field [0001] The invention belongs to the technical field of medicine and relates to a sperm quality evaluation method based on flow cytometry. Background technique [0002] Sperm quality assessment is an important indicator of male fertility. The traditional sperm quality assessment technology is to use a phase contrast microscope and a special blood cell counting panel to observe with the naked eye, mainly to evaluate and analyze the sperm count, sperm motility and sperm morphology. In the past two decades, with the continuous advancement of computer technology, special analysis software combined with high-resolution image acquisition devices has appeared clinically to evaluate and analyze sperm quality. The main evaluation indicators are the same as the traditional sperm quality evaluation methods. Although the computer-aided sperm quality assessment method (CASA) reduces the work intensity of visual assessment, especially the assessment of sperm motility is m...

Claims

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Application Information

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IPC IPC(8): G01N15/14
Inventor 胡勇华黄春波温韵洁王晓丹
Owner 广州华银医学检验中心有限公司
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