The Working Principle and Application of Flow Cytometer (I)

Flow cytometry (FCM) is a high-scientific technology developed in the 1970s. It began to develop from basic research to clinical medical research and disease diagnosis and treatment monitoring in the 1980s.

It combines optics, electronics, fluid mechanics, cytochemistry, biology, immunology, laser and computer and other disciplines and technologies into one, has the function of analyzing and sorting cells at the same time, and can quickly analyze the cell suspension. The cells arranged in a single row in the flowing liquid are tested one by one to obtain the light scattering and fluorescence indexes of the cells.

It can not only measure cell size, internal particle properties, but also detect cell surface and cytoplasmic antigens, intracellular DNA, RNA content, etc., can analyze population cells at the single cell level, detect and analyze a large number of cells in a short time, and collect, store and process data for multi-parameter quantitative analysis; it is also able to collect (sort) cells of a certain subgroup with sorting purity> 95%.

Flow cytometry (FCM) is widely used in hematology, immunology, oncology, pharmacology, molecular biology and other disciplines. It has the characteristics of fast detection speed, many measurement indexes, large amount of collected data, comprehensive analysis and flexible methods.

Scientific research flow cytometers (such as FACStar, FACSort, EPICS-Elite, etc.) have complete functions and flexible analysis, but the operation is more cumbersome and must be operated by professional technicians who have been formally trained. Clinical instruments (FACScan, Profile, etc.) are easy to operate, have good stability and fast analysis speed, and are suitable for application in clinical laboratories.

With the continuous improvement of the performance of the instrument, new types of flow cytometers with simple operation have come out one after another. The continuous discovery of new reagents has reduced the cost of testing, and FCM has gradually entered the clinical laboratory from the research room, becoming an important means of routine experimental diagnosis, which not only provides an important diagnostic basis for the clinic, but also makes the diagnostic level and experimental technology of the laboratory department raise to a new height.

The working principle of flow cytometry

The working principle of the flow cytometer is that the cells to be tested are stained with specific fluorescent dyes and placed in a sample tube, and enter the flow chamber filled with sheath fluid under the pressure of the gas. The cells are arranged in a single row under the constraint of the sheath fluid and are ejected from the nozzles of the flow chamber to form a cell column.

Flow cytometry usually uses laser as the light source. After focusing and shaping, the beam is irradiated vertically on the sample stream, and the fluorescently stained cells produce scattered light and excited fluorescence under the irradiation of the laser beam.

These two signals are simultaneously received by the forward photodiode and the photomultiplier tube in the 90 ° direction. The light scattering signal is detected at a small forward angle. This signal basically reflects the size of the cell volume; the reception direction of the fluorescent signal is perpendicular to the laser beam, and is separated by a series of dichroic mirrors and band pass filters to form multiple fluorescent signals with different wavelengths.

The intensity of these fluorescent signals represents the intensity of the measured cell membrane surface antigen or the concentration of its nuclear substance, which can be converted into an electrical signal after being received by the photomultiplier tube, and then through the analog-to-digital converter, the continuous electrical signal is converted into a digital signal recognized by a computer.

The computer processes the measured signals and displays the analysis results on the computer screen, which can also be printed out. It can also be stored on the hard disk in the form of a data file for future inquiry or further analysis.

The display of detection data can be selected in various forms depending on the measurement parameters. The single-parameter data is expressed in the form of a histogram, where the X axis is the measured intensity and the Y axis is the number of cells.

In general, the resolution of the coordinate axis of the flow cytometer has 512 or 1024 channels, which depends on the resolution of its analog-to-digital converter. For dual-parameter or multi-parameter data, you can display the histogram of each parameter separately, or you can choose a two-dimensional three-point graph, contour map, grayscale graph or three-dimensional stereoscopic view.

Cell sorting is achieved by separating droplets containing single cells. An ultra-high frequency transistor is equipped on the nozzle of the flow chamber. After charging, it vibrates to break the sprayed liquid into uniform droplets. The cells

Charge these droplets with different positive and negative charges. When the droplets flow through a deflection plate with several thousand volts, they are deflected under the action of a high-voltage electric field and fall into their respective collection containers. The uncharged droplets fall. Into the middle waste container to achieve cell separation. The sorted cells can be cultured or treated for further research.

Clinical application of flow cytometry

With the deepening of FCM research, its value has entered the stage of clinical application from scientific research and has been widely used in the field of clinical medicine.

1. The role in clinical cellular immunity

FCM analyzes cell surface antigens by fluorescent antigen antibody detection technology, and performs cell classification and subpopulation analysis. This technology plays an important role in the evaluation of human cellular immune function and the diagnosis and treatment of various hematological diseases and tumors. There are a large number of articles on the changes of lymphocyte subsets in various diseases.

The T4 / T8 ratio of lymphocytes in normal population is about 2: 1, but the ratio may be reversed when the immunity of human cells is low. FCM can also monitor the renal rejection of patients after kidney transplantation. If the T4 / T8 ratio is inverted, the patient has a good prognosis and fewer renal rejections occur; otherwise, the risk of rejection increases.

The same measurement technique is also used in the diagnosis and treatment of AIDS. The authors also reported the reference value of peripheral blood lymphocyte immune phenotype, and discussed the influence factors such as race, gender, and age.

To be continued in Part II…