Acute lymphoblastic leukemia (ALL) is a type of cancer caused by the disordered growth of white blood cells, known as lymphomas, which are formed in the bone marrow and rapidly diffuse through the bloodstream; affecting other organs and eventually leading to death. Diagnostic tests currently implemented require a cytometric analysis of bone marrow extraction or peripheral blood marking for counting of blastic cells present in peripheral blood via light microscopy. These techniques are invasive, requiring labeling and alteration of cells. Therefore, an alternative technique is sought by implementing a diffraction phase microscope (DPM), which allows the measurement of the size and refractive index variations of the cells in a non-invasive way, looking for the detection of blast cells in human peripheral blood. In a first phase it is necessary to distinguish blastic cells from other mononuclear cells such as T and B lymphocytes and monocytes. The present work describes the implementation of the technique in order to establish parameters of population differentiation, morphometric and refractive index of mononuclear cells and blast cells in a single blood sample. For this purpose it is described the process of separation of peripheral blood mononuclear cell populations and cells from two diseased donors are analyzed. Consequently, the DPM technique is validated as a differentiation parameter, opening the way to the possibility of validating it for the diagnosis of ALL in the analysis of a sample of human peripheral blood.