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An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio.

ABSTRACT: The nucleus-to-cytoplasm ratio (N:C) can be used as one metric in histology for grading certain types of tumor malignancy. Current N:C assessment techniques are time-consuming and low throughput. Thus, in high-throughput clinical contexts, there is a need for a technique that can assess cell malignancy rapidly. In this study, we assess the N:C ratio of four different malignant cell lines (OCI-AML-5-blood cancer, CAKI-2-kidney cancer, HT-29-colon cancer, SK-BR-3-breast cancer) and a non-malignant cell line (MCF-10A -breast epithelium) using an imaging flow cytometer (IFC). Cells were stained with the DRAQ-5 nuclear dye to stain the cell nucleus. An Amnis ImageStreamX® IFC acquired brightfield/fluorescence images of cells and their nuclei, respectively. Masking and gating techniques were used to obtain the cell and nucleus diameters for 5284 OCI-AML-5 cells, 1096 CAKI-2 cells, 6302 HT-29 cells, 3159 SK-BR-3 cells, and 1109 MCF-10A cells. The N:C ratio was calculated as the ratio of the nucleus diameter to the total cell diameter. The average cell and nucleus diameters from IFC were 12.3 ± 1.2 μm and 9.0 ± 1.1 μm for OCI-AML5 cells, 24.5 ± 2.6 μm and 15.6 ± 2.1 μm for CAKI-2 cells, 16.2 ± 1.8 μm and 11.2 ± 1.3 μm for HT-29 cells, 18.0 ± 3.7 μm and 12.5 ± 2.1 μm for SK-BR-3 cells, and 19.4 ± 2.2 μm and 10.1 ± 1.8 μm for MCF-10A cells. Here we show a general N:C ratio of ~0.6-0.7 across varying malignant cell lines and a N:C ratio of ~0.5 for a non-malignant cell line. This study demonstrates the use of IFC to assess the N:C ratio of cancerous and non-cancerous cells, and the promise of its use in clinically relevant high-throughput detection scenarios to supplement current workflows used for cancer cell grading.

SUBMITTER: Sebastian JA

PROVIDER: S-EPMC8224973 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundImage segmentation is the process of partitioning an image into separate objects or regions. It is an essential step in image processing to segment the regions of interest for further processing. We propose a method for segmenting the nuclei and cytoplasms from white blood cells (WBCs).MethodsInitially, the method computes an initial value based on the minimum and maximum values of the input image. Then, a histogram of the input image is computed and approximated to obtain function values. The method searches for the first local maximum and local minimum from the approximated function values in the order of increasing of knots sequence. We approximate the required threshold from the first local minimum and the computed initial value based on defined conditions. The threshold is applied to the input image to binarize it, and then post-processing is performed to obtain the final segmented nucleus. We segment the whole WBC before segmenting the cytoplasm depending on the complexity of the objects in the image. For WBCs that are well separated from red blood cells (RBCs), n thresholds are generated and then produce n thresholded images. Then, a standard Otsu method is used to binarize the average of the produced images. Morphological operations are applied on the binarized image, and then a single-pixel point from the segmented nucleus is used to segment the WBC. For images in which RBCs touch the WBCs, we segment the whole WBC using SLIC and watershed methods. The cytoplasm is obtained by subtracting the segmented nucleus from the segmented WBC.ResultsThe method is tested on two different public data sets and the results are compared to the state of art methods. The performance analysis shows that the proposed method segments the nucleus and cytoplasm well.ConclusionWe propose a method for nucleus and cytoplasm segmentation based on the local minima of the approximated function values from the image's histogram. The method has demonstrated its utility in segmenting nuclei, WBCs, and cytoplasm, and the results are satisfactory.

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An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio.

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