The Coulter LH750 system measures the count and size of leukocytes, erythrocytes and PLTs using the new AccuCount method, that integrates the Coulter impedance principle with new technologies and software algorithms with histogram distributions. In the flow-cell of the RBC/PLT channel, all electrical pulses with volume between 36 and 360 fL that are considered valid by the “Pulse Edit” system are classified as RBCs and distributed in 256 channels with a volumetric resolution of 1.3 fL per channel. From the RBC histogram the system determines the parameters MCV and RDW (Red cell Distribution Width): this last expresses the degree of heterogeneity of the RBC population volume, and is therefore an index of erythrocyte anisocytosis.

All pulses with volume between 2 and 20 fL are classified as PLTs and distributed in 256 volumetric channels; MPV and PDW indices are derived from the histogram thus obtained. Leukocyte count is also carried out with the impedance method, after erythrocyte lysis. It includes all particles with a volume greater than 35 fL. An accurate control of possible interferences is obtained through electronic analysis of electrical pulses. In particular, a WBC histogram is obtained for each of three counting apertures. In the volume channel cells are subdivided into 256 channels on the abscissa and the relative frequency of cells in ordinate: thus three distinct leukocyte populations of lymphocytes, neutrophils and medium-sized cells are defined. The determination of the leukocyte differential count is based on the VCS technology, that provides a triple simultaneous measurement of cell volume (DC), electrical conductivity (RF) and scatter (diffraction of laser light). Each cell then produces three specific response signals, which are analyzed by an electronic circuit to define the type of cell population it belongs to, as well as the presence of abnormal cell distributions. The classic impedance method, that measures the opposition produced by cells to the passage of a direct continuous low-frequency current (DC), is used to determine cell volume: variations of voltage produced by the passage of leukocytes throughout the aperture is recorded by the electronic circuit in the form of a pulse, whose amplitude is directly proportional to the volume of the cell. The measure of conductivity is obtained using a high (radio) frequency current (RF) of 23 MHz, that passes through the cell and produces a RF signal related to cell conductivity, or resistance of the internal cell content to the current flow. The cellular property thus analyzed is the reciprocal of conductivity: it is defined opacity. Among the internal structural elements of the cell, the nucleus has the lowest resistance and is therefore a good conductor of current. Cells with a high nucleo-cytoplasmic ratio (lymphocytes ) do possess minor opacity than cells with reduced nucleo-cytoplasmic ratio (granulocytes ). Finally, the presence of granules within the cytoplasm is detected through modifications of the beam of monochromatic laser light, emitted at 655 nm from a helium-neon laser, when it hits every cell. A multiangle detector at 10-70 ° is used to evaluate the light scattered from each cell at different angles: these measurements are directly proportional to their granularity, reflectance and membrane structure. Thus the combined measurement of laser light scatter, impedance and conductivity/opacity allows a complete classification of leukocyte populations based on specific “morphological” characteristics: cell volume, nuclear size and density, nucleo-cytoplasmic ratio, cytoplasmic granules and cell surface.

The Coulter systems, in addition, provide in all analyzed samples 24 new positional parameters for the main leukocyte populations and reticulocytes: mean value (MEAN) and degree of heterogeneity (SD) of the measures of cell volume, opacity/conductivity (related to nuclear density and nucleo-cytoplasmic ratio), and scatter of laser light (related to internal granularity and cell surface characteristics). These positional parameters, or CPD (Cell Population Data), permit an objective and numerical assessment of morphological characteristics of cell populations, on the basis of their position within the distribution clusters in the three-dimensional graphics. VCS method is also used for a qualitative and quantitative analysis of reticulocytes and erythroblasts. Reticulocytes are counted after automatic staining with new methylene blue. In addition to the total absolute and proportional counts, an immature reticulocyte fraction (IRF) and the mean volume of reticulocytes (MRV) are also calculated. A complex algorithm determines the count of erythroblasts at the same time of the leukocyte differential count.