Fully Automated Fluorescence Compensation Method for Flow Cytometry

Abstract

The invention discloses a fully automatic fluorescence compensation method for a flow cytometer, comprising the following steps: step 1) preparing a multi-color sample and testing it on a machine to obtain the original fluorescence signal result; step 2) maintaining the voltage gain and step 1) In the same way, each single-color bead and blank bead are mixed in proportion to be tested on the computer, and the original output value of the fluorescence signal of each single-color bead and blank bead subset in each detection channel is obtained; step 3) calculate the fluorescence leakage Matrix K and autofluorescence matrix A; step 4) calculate fluorescence compensation matrix K C ; Step 5) perform automatic fluorescence compensation to obtain the detection result after fluorescence compensation. The present invention utilizes mixed balls, and only needs to test once to obtain the compensation result, without the need to test the negative control samples and each single positive sample multiple times, which simplifies the operation, shortens the time, and eliminates the error between experiments; the present invention also The specific calculation method of the fluorescence leakage matrix K is disclosed, and the method of the invention is simple and convenient, and the effect is remarkable.

Description

technical field [0001] The invention relates to the field of flow cytometry data analysis, in particular to a fully automatic fluorescence compensation method for flow cytometry. Background technique [0002] When performing flow cytometry analysis, the sample to be tested usually carries two or more fluoresceins, such as phycoerythrin (PE), fluorescein isothiocyanate (FITC), and silk fibroin (PerCP), etc. After being excited by laser, fluorescein emits fluorescence of different wavelengths. Theoretically speaking, different fluorescent lights can be separated by selecting appropriate spectroscopic components, so that each detector only receives one fluorescent signal and does not detect other fluorescent signals. However, in fact, the excitation wavelengths or emission wavelengths of commonly used fluorescent dyes are normally or skewed, and have a wide range. Therefore, although their emission peaks are different, the emission spectra often overlap, such as figure 1 As s...

AI Technical Summary

Problems solved by technology

Manual compensation can complete the compensation function well for two-color and three-color analysis, but for four-color and more than four-color analysis, the operation is so difficult that it is almost impossible to realize

However, the automatic compensation method provided by the flow cytometer needs to prepare negative control samples and all single positive control samples, which requires a large amount of samples, tedious sample preparation, and many sample detection times, resulting in high difficulty in implementation

And due to the error between multiple detections, the compensation effect is often not good

Images