The Cell Dyn blood cell counters basically exploit a twofold methodological approach: electrical impedance and optical measurement. They count cells by electrical impedance and classify leukocytes by measuring the diffusion of a laser light beam at three different angles: 0 °, proportional to cell size; 10 °, proportional to the structure; 90 °, proportional to cytoplasmic granularity. Furthermore, eosinophils are separately classified thanks to the specific capacity of their granules for depolarizing polarized light. The Cell Dyn 4000 system was the first, among the automated hematologic analyzers, to use an argon laser similar to those of non-dedicated flow cytometers. This approach made possible new applications in fluorescence, such as direct counting of reticulocytes and erythroblasts, and measurement of cell viability. As for erythrocytes measurement, these cells are subjected to a process of sphering by chemical reagents, aimed at minimizing the impact on MCV of changes of cell shape and viscosity. The analysis of reticulocytes is based on the use of a new fluorescent dye (CD4K530 ), which brightly stains nucleic acids (RNA and DNA): this allows a clear separation of reticulocytes, that contain small amounts of RNA, from unstained mature erythrocytes, as well as from nucleated erythroid cells, that contain larger quantities of nuclear DNA. An assessment of the maturity of reticulocytes is also obtained thanks to their higher concentration of RNA. After automated dilution, incubation and staining of the blood sample, reticulocytes pass through the flow cell, where light scattered at intermediate angle (7°) and green fluorescence (FL1, 530 nm) are measured. PLTs are excluded because of their lower scatter at 7°. Nucleated cells (leukocytes and erythroblasts) are excluded owing to the higher fluorescence of DNA in their nuclei. PLTs are measured using both impedance ( PLTi ) and a two-dimensional optical method (PLTo ), based on the detection of light scattered at 90° and 7° angles. The impedance PLT count has a function of quality control of the main optical counts and is reported only when the difference between the two counts is significant. It is also possible to perform an immunological PLT counts (with anti- CD61 specific monoclonal antibodies): this is useful for clinically important samples with suspect interferences, as well as for critical thrombocytopenic counts.

As far as leukocyte count is concerned, the analysis is carried out in the optical channel: a segment of the sample is diluted, hemolyzed and stained with the DNA-specific dye propidium iodide (PI), which emits red fluorescence when hit by laser light. Following the lysis of cell membranes, nuclei of erythroblasts are rapidly stained by PI, which also binds to any erythrocyte inclusions that contain nucleic acid, such as Howell- Jolly bodies and malaria parasites. PI selectively penetrates into leukocytes as a consequence of the lolss of integrity of their membranes: it cannot get into intact cells, while it quickly stains nuclear DNA in damaged or non- viable cells whose membranes have become permeable. Within the flow cell, stained leukocytes are subjected to a complex detection of light scattered at three different diffraction angles, as well as to the measurement of cell fluorescence and scatter of depolarized light. Eosinophils are separated from neutrophils on the basis of their greater ability to change the plane of polarization of the laser beam (depolarization). Full differential count cytograms are built using only two dimensions (XY) at a time. A different color is used in the cytogram to specifically classify each cellular cluster. The red fluorescence (FL3), originated after staining with PI, is used to identify erythroblasts (nucleated RBCs or NRBCs), defined by three parameters: axial light loss (ALL at 0 °), intermediate angle scatter (IAS at 7°) and red fluorescence (FL3). On the basis of these properties, NRBCs are separated from intact leukocytes as well as from non-vital cells, forming a cluster of discrete signals that allows their count (both in absolute number and as a number per 100 leukocytes). The remaining highly fluorescent cells are classified as non-viable leukocytes and their number is expressed as a WBC viability fraction (WVF).

Blasts, immature granulocytes, band cells and atypical lymphocytes are identified on the basis of a weighted multidimensional assessment of the variation degree of cluster position and dispersion, in comparison to mature normal cell populations. Finally, a count in automation of CD4+ and CD8+ cells, with CD4/CD8 ratio calculation, is also carried out: this is useful for an easy and quick screening of samples with abnormal lymphocyte subclasses.