Principles of fluorescence flow cytometry and somatic cell counting
To measure the concentration of somatic cells in a sample, the cells must be stained or labeled with a fluorescent dye. These fluorescent molecules, commonly known by the chemical name DAPI (4’, 6-Diamidine-2’-phenylindole dihydrocloride) are absorbed by the nuclear DNA of the somatic cell.
The cells are then individually illuminated by light of a defined wavelength (UV). This light activates the fluorescent molecules to emit light of a specific color or wavelength: DAPI emits the color yellow. The intensity of fluorescence emitted from each cell indicates the quantity of cell substance contained.
Cell concentration determination:
Each fluorescent cell in this volume is counted. From that, the concentration of cells per ml is determined. The light intensity, which represents the cell substance of interest, is displayed on the horizontal axis of a histogram; the number of cells is displayed on the vertical axis. Light intensity is related to the size of cells—the further to the right on the horizontal axis, the higher the light intensity and the bigger the size of the cell. On the vertical axis, the higher the peak, the more counts of a certain size of cells.
Sample illumination and measurment in the Delta Instruments’ SomaScope™ Flow Cell:
The principle of counting is to pass the cells in the liquid one by one through a cell, where they are illuminated by the excitation light and the fluorescent light is detected.
The flow cell has three connections. The first is for the sample input, the second for the sheath fluid input and the third for the waste output. The sample flows through a capillary into the flow cell. Here, the sheath fluid takes it with it. Because of the specific flow cell geometry, the sheath and sample current speed up into a very narrow, laminar-flowing sample stream. The sample stream is not mixed with the sheath stream. The cells labeled with fluorescent dye, pass the measuring area, one after the other. The excitation light individually illuminates the cells and the fluorescent light is measured and analyzed.
Delta Instruments’ SomaScope Smart uses two different photo-multipliers for detection. This enables a clearer separation of noise signals from somatic cells. Somatic cells passing the flow cell produce light pulses, which are detected by both photo-multipliers. The actual count is a result of a differentiation between both photo-multipliers. Only those pulses that are simultaneously detected on both photo-multipliers are counted. A percentage of the match between both photo-multipliers is displayed together with the count result. This percentage is called the Q-value (quality of the measurement). The Q-value is a variable for the operator to see if performance of the instrument and the condition of the samples are good. The Q-value on good condition samples and with a well-adjusted instrument is higher than 90 percent.