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.
The Digital Holographic Microscopy in Transmission technique (DHM) is considered a useful tool in the noninvasive quantifying of transparent biological objects like living cells. In this work, we propose this technique to study and to monitor control macrophages infected by Leishmania (mouse lineJ774.A1). When the promastigotes enter in contact with healthy macrophages, they got phagocytosed and latterly confined in the formed parasitophorous vacuole. These processes change the morphology and density of the host macrophage. Both parameters can be measured in a label-free analysis of cells with the aid of the DHM technique. Our technique begins with the optical record of the holograms using a modified Mach-Zehnder interferometer and the reconstruction of the complex optical field transmitted by macrophages. In the latter point, we employ the angular spectrum algorithm. With the complex optical field reconstruction, we compute the field amplitude and the phase difference maps, which leads to describe one morphological characterization for the samples. Using phase difference maps is possible to measure internal variations for the integral refractive index, estimating the infection level of macrophages. Through the changes in the integral refractive index, it is also possible to describe and quantify in two different states the evolution of the infection. With these results some parameters of cells have been quantified, making the DHM technique a viable tool for diagnosis of biological samples under the presence of some pathogen.