Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm

Abstract

Background Sperm DNA integrity is of paramount importance in the prognosis of fertility. We applied image cytometry to a toluidine blue (TB) test we recently proposed. Methods Sperm samples from 33 men were assayed for standard sperm parameters and classified as normal or abnormal. Sperm smears were subjected to the TB test, DNA denaturation testing with acridine orange (AO), and terminal deoxyuridine triphosphate biotin nick end labeling (TUNEL). In CCD image analysis, TB‐stained sperm cell heads were microscopically assigned to one of four color groups (dark, blue, light violet, and light blue). The optical densities of 6,600 cells in green and red CCD images were used to elaborate an algorithm for discrimination of these groups. Results The proportions of sperm in TB color groups, as estimated with the developed image cytometry algorithm, correlated with microscopic features. The number of TB dark cells correlated with the number of AO‐red and TUNEL+ cells. The proportion of TB dark cells in normal samples did not exceed 35%. Light‐blue sperm cell heads prevailed in normal samples, whereas dark and blue sperm cell heads dominated in abnormal samples. Conclusions The TB test was suitable for the assessment of sperm cell DNA integrity. The elaborated image cytometry algorithm can be used for this purpose and for finer determination of sperm nucleus status. Cytometry Part A 52A:19–27, 2003. © 2003 Wiley‐Liss, Inc.

Evaluación de la calidad del ADN espermático en camélidos sudamericanos y otras especies domésticas

“…Esto permite, utilizar al DTT o al ME como controles positivos de la tinción con AT. Esta tinción se ha utilizado en varias especies para detectar defectos en la condensación de la cromatina (humano: Barrera et al, 1993;Erenpreiss et al, 2001;Erenpreisa et al, 2003;Tsarev et al, 2009;toro: Beletti y Mello, 1996;Beletti et al, 2005;conejo: Beletti y Mello, 2004;padrillo equino: Naves et al, 2004;Sardoy et al, 2008;Carretero et al, 2012a) y actualmente se ha empleado para evaluar la cromatina en caninos, felinos y porcinos (Monachesi y Carretero, 2014;Allera et al, 2016;Arraztoa et al, 2016).…”

“…En nuestro laboratorio, se probó simplificar la técnica descripta por otros autores (Erenpreiss et al, 2001(Erenpreiss et al, , 2004Erenpreisa et al, 2003;Beletti y Mello, 2004;Beletti et al, 2005;Tsarev et al, 2009), quienes utilizaron una hidrólisis ácida previa a la tinción con la finalidad de aumentar la sensibilidad de la técnica. Estos autores reportaron que los espermatozoides que tienen una cromatina altamente compacta (normales) son apenas afectados por la hidrólisis ácida y en consecuencia se tiñen levemente celestes, mientras que, en los espermatozoides con una cromatina alterada, las protaminas pueden ser parcialmente extraídas por la hidrólisis permitiendo que las moléculas de AT se unan a los grupos fosfato, aumentando la sensibilidad de la técnica.…”

Supraphysiological Free Radical Levels and their Pathogenesis in Male Infertility

“…Toluidine Blue (TB) test [131,132]. The test measures the availability of the sperm chromatin DNA phosphate residues for staining with TB, which is dependent on both the protein state and DNA integrity.…”

“…The test measures the availability of the sperm chromatin DNA phosphate residues for staining with TB, which is dependent on both the protein state and DNA integrity. Four cell groups with different optical densities can be distinguished by the TB test [132]. These correspond to the following visual TB colours: dark violet cells (TBDCs; abnormal chromatin structure), light blue cells (TBLCs; normal chromatin structure) and two intermediate forms: light violet and dark blue (probably with less damaged chromatin structure).…”

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis

“…Quantitative luminescence measurements of biomolecules, single cells and tissue specimens in solid phase are particularly valuable for identification and unambiguous confirmation of rare cell types [1][2][3] , time-lapse study of live cells [4][5][6] , profiling of subcellular components and biomolecular expressions [7][8][9] , and a broad range of other diagnostics applications [10][11][12] . The existing techniques based on digital microscopy [13][14][15][16] , however, are time-consuming and resource-demanding, as images are typically captured for the entire sample area, or even through three-dimensional space [17][18][19][20] , followed by stitching and processing to identify and quantitate targets of interest.…”