The technology of the Siemens ADVIA system represents the most recent development and improvement of automated flow cytochemistry, a method firstly introduced in 1975. The system includes four analytical channels, respectively dedicated to measurements of hemoglobin concentration, RBCs and PLTs, WBC total and differential count after cytochemical staining for myeloperoxidase, and count of basophil granulocytes in association with density measurement of the nuclei of the other leukocytes. In each channel, cells are diluted and modified by the action of specific reagents and subsequently analyzed through the detection of the modifications that the impact with every single cell produces in a beam of incident light. These data are processed and classified using complex algorithms, from which numbers and cell distributions (histograms and cytograms) are derived. The peroxidase channel, in particular, is used to obtain the total and differential WBC count, except the count of basophils, here included in the category of lymphocytes. In addition to four classes of normal cells, the peroxidase channel provides the percentage of large unstained (i.e., peroxidase-negative) cells, or LUC: they correspond, when they exceed a threshold value of 4%, to atypical leucocytes that require microscope verification. The MPXI index indicates the average peroxidase activity of the neutrophil population in each sample. The basophil/nuclear density channel uses laser light and an optical system of detection: pretreatment of the sample in the reaction chamber causes lysis of RBCs and rupture of the cytoplasmic membranes of leukocytes, which lose cytoplasm and its contents. Basophils are resistant to the action of the reagent and can be identified by virtue of their preserved size; the nuclei of all other leukocytes are collected in the lower part of the cytogram and form two contiguous clusters of mononuclear cells ( MN) and polymorphonuclear cells (PMN). The ratio between the modal values of PMN and MN clusters is expressed and reported as a lobularity index (LI), which gives an indication of the degree of nuclear segmentation of neutrophils; an alarm of left shift is triggered when hyposegmentation and band cells are detected. Blast cells have a hypodense chromatin structure: this feature results in low wide angle scatter signal and localization of immature nuclei in the left lower side of MN cells cluster. The ADVIA system compares for each sample the sum of granulocytes (neutrophils + eosinophils) in the peroxidase channel with the percentage of PMN in the basophil channel: a discrepancy of more than 7.5 % leads to an immature granulocytes (IG) flag.

The ADVIA channel for the counting of RBCs and PLTs uses the same optical system as the basophil channel, with dual-angle laser light detection: each erythrocyte is thus characterized by a double signal, from which the system calculates the MCV, as well as a direct measure of hemoglobin concentration (CHCM). As an internal control, CHCM is compared with the calculated MCHC value, corresponding to the ratio between hemoglobin and hematocrit. The same method is applied for the analysis of reticulocytes, using the specific dye oxazine 750. The set of measurements is represented graphically in the form of histograms and a two-dimensional cytogram of volume versus hemoglobin concentration. This method can precisely calculate the percentage of RBCs that are microcytic (MCV<60 fL) and hypochromic (CHCM <28 g/dL), as well as the immature reticulocyte fraction (IRF) and reticulocyte indices of volume (MCVr) and hemoglobin content (CHr). PLT distribution is visible in the form of both a histogram and a two-dimensional cytogram that collects from the two different scatter angles all signals in the volume range between 0 and 30 fL. Thus PLTs of normal size are separated from large PLTs, microcytic RBCs, erythrocyte fragments and empty stroma.

Finally, the integration of signals from different analytical channels permits the identification and counting of circulating erythroblasts.