Evaluating Confocal Microscopy as a Tool to Diagnose Red Blood Cell Diseases

Hemoglobinopathies represent the most common single-gene defects in the world and pose a major public health problem. These defects can affect the synthesis of hemoglobin, the molecule in charge of oxygen transportation, quantitatively (thalassemia) or qualitatively (sickle cell disease). These affectations will terminally lead to the premature destruction of red blood cells (RBCs), nevertheless they have different etiologies and they need to be treated differently. Diagnosing a patient can sometimes be difficult due to the coexistence of several of such diseases and/or blood transfusions, and requires expensive and complex molecular tests. But alternatively, it is well-known that the spectral information of RBC autofluorescence and immunostaining can reveal important differences among these RBCs diseases [1] – [3] . The isolated assessment of cell shape has been proven to be insufficient. In these context, we conducted a first pilot study to analyze the spectral and morphological characteristics of healthy and diseased RBCs, under a spectral confocal microscope Leica TCS SP8 (Leica Microsystems GmbH, Mannheim, Germany). Specifically, fresh blood samples from 17 pediatric patients ages 1 to 17, some suffering from α-thalassemia and iron deficiency were evaluated. Autofluorescence was captured in the 425-790 nm range, using hybrid detection that provides with a higher sensitivity, a 63x (NA 1.4, oil) plan-apochromatic objective, and a diode laser line with emission at 405 nm for excitation. The autofluorescence signals captured showed spectral differences that can be used to discriminate among diseases and severity, especially at the 628 nm band as showed in Figure 1 .