We have used five independent variables on a flow cytometer to discriminate and to quantify the cellular components within both blood and bone marrow aspirates. The signals were stored in list mode by which a five-dimensional space was created. The cells–differentiated into: (1) erythrocytes, (2) reticulocytes, (3) nucleated erythroid cells, (4) platelets, (5) lymphocytes, (6) monocytes, (7) neutrophils, (8) eosinophils, and (9) immature leukocytes–had to meet unique criteria with regard to their characteristics in the created five-dimensional space in order to be classified in a specific cell category.
Forward and orthogonal light-scattering signals were matched with three fluorescence variables to obtain discrimination without necessitating erythrocyte lysis.
Thiazole orange (binding predominantly to RNA) and LDS-751 (principally detecting DNA) were used to differentiate erythrocytes, platelets, reticulocytes, and nucleated cells. A monoclonal antibody, CD45, conjugated with phycoerythrin, was used to aid in discriminating between lineages of nucleated cells.