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Cytological and Biomolecular Measuring Procedures

Working Group 8.32

Principle of cell counting

Introduction

Cells can readily be counted using a microscope. The exact sample volume is usually determined using counting chambers, which have a known height. The sample volume is then derived using the calibrated microscope magnification. However, detailed morphological information is usually not needed for proper identification of cell type. In this case flow cytometry offers advantages over microscopic counting for examination of cell suspensions. A large number of cells can be measured by flow cytometry. This is essential to measure the concentration of rare cells in a mixture (e.g. stem cells circulating in blood). In addition the concentration can be measured with lower statistical uncertainty. Two detection techniques are routinely applied in flow cytometry, laser flow cytometry and impedance counting.

Principle of laser flow cytometry

Blood cells of a diluted measurement suspension are injected into a capillary, and hydrodynamically focussed by a sheath flow to finally enter the flow channel (250 µm x 250 µm) of a quartz cell. The diameter of the sample fluid, which contains the blood cells, is reduced to about 5 µm because of hydrodynamic focussing. Hence the blood cells cross the focus of the laser beam in single file. Intensity of forward light scatter and orthogonal light scatter is measured simultaneously for each blood cell. In addition, laser-induced fluorescence signals are detected when analysing stained blood cells.

In the figure on the right hand side the result from a measurement of a diluted blood sample is shown. The relative intensity of forward light scatter at  a wavelength of 632.8 nm is plotted versus forward light scatter at 413.1 nm. In this scatter diagram, each blood cell is represented by a single dot. The clusters indicated in the scatter diagram correspond to red blood cells (RBC), platelets (thrombocytes, T) and subpopulations of leukocytes, i.e. lymphocytes (Ly), monocytes (M) and granulocytes (G).

The axes represent the integrated differential cross section for light scattering, the corresponding angle of observation ranges from 3.3° to 17.4°. For calibration of the cross section monodisperse polystyrene microspheres of known sizes can be used, the integrated cross sections of which are calculated by Mie theory.

Principle of impedance counting

Blood cells of a diluted blood sample pass a measuring sensor in single file. Typically, the diameter and length of the orifice amounts to 60 µm. The front sheath fluid is used for hydrodynamic focussing of the sample flow, which contains the blood cells. The rear sheath flow is required to avoid recirculation of blood cells after passing the orifice. As sheath fluids, isotonic solution is used. A constant current of up to 1mA is applied between the electrodes. The voltage between both electrodes is changed due to the modified electrical conductivity when a blood cell passes the measuring sensor.

This effect allows detection of single blood cells. The amplitude of the signal is approximately proportional to the particle volume and hence red blood cells (volume about 90 fL) and platelets (volume about 6 fL) can be distinguished. A typical histogram is shown in the figure to the left. Here the number of events is plotted in a histogram, i.e. cell count versus the amplitude of the impedance signal. Two distributions of cells are observed corresponding to red blood cells (erythrocytes) and platelets (thrombocytes).

To enumerate leukocytes by impedance counting erythrocytes are destroyed by lysing reagents. Subsequently the measuring suspension is analysed. A typical resulting pulse height distribution of a control blood sample is shown in the histogram on the right hand side.

Flow cytometric cell sorting

Blood cells of a diluted measurement suspension are injected through a hollow needle and hydrodynamically focussed by a sheath flow. Sample and sheath flow pass a nozzle (diameter 50 µm -  100 µm) resulting in a free liquid jet in air. The blood cells cross the laser beams in single file and are analyzed as usual in optical flow cytometry by observing  simultaneously forward light scatter, side scatter and laser-induced fluorescence.

For sorting of single cells the liquid jet is broken up into small droplets by piezoelectrical modulation of the whole assembly. The sheath fluid is isotonic salt solution, which is electrical conductive. The droplet that contains the selected cell is charged, deflected in a static electrostatic field and collected in a tube. Alternatively, selected cells are sorted onto slides for subsequent microscopic investigations, for example to attribute cells to a specific subpolulation.