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Minimizing false positives for CTC identification
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Resumo(s)
Background: Cancer is a leading cause of death worldwide, with metastasis playing a significant role. Circulating Tumour Cells (CTCs) can provide important real-time insights into tumour heterogeneity and clonal evolution, making them an important tool for early diagnosis and patient monitoring. Isolated CTCs are typically identified by immunocytochemistry using positive biomarkers (cytokeratin) and exclusion biomarkers (CD45). However, some white blood cell (WBC) populations can express low levels of CD45 and stain non-specifically for cytokeratin, increasing their risk of misclassification as CTCs. There is a clear need to improve CTC detection and enumeration criteria to unequivocally eliminate interfering WBC populations. Results: This study showed that, indeed, some granulocyte subpopulations expressed low levels of CD45 and stained non-specifically for cytokeratin, misidentifying them as CTCs. These same cells, however, strongly expressed CD15, allowing them to be identified as WBCs and excluded from CTC classification. Flow cytometry confirmed the specificity of the CD15 antibody for the granulocyte subpopulation. False positives were considerably reduced from 25 % to 0.2 % by double exclusion, combining a CD15 antibody with a highly specific CD45 antibody. Furthermore, complete elimination of potential false positives was achieved using double exclusion in combination with improved selection of cytokeratin antibody. The study emphasises the importance of a robust exclusion criteria and high antibody specificity in CTC immuno-assays for accurate identification of CTC candidates and thorough exclusion of interfering WBC subpopulations. Significance: This study demonstrated how misidentifying a granulocyte subpopulation can lead to inaccurate CTC evaluation. However, sensitivity and specificity of CTC identification may be improved by using high-performing antibodies and by including a second exclusion biomarker, in turn, allowing for a more comprehensive clinical application of CTCs.
Descrição
Funding Information: This work was supported by PROMISE project funded through the Caixa Health Research program from La Caixa Foundation (HR20-00637); and Health From Portugal (C630926586-00465198), supported by Component C5 – Capitalisation and Business Innovation, under the Portuguese Resilience and Recovery Plan, through the NextGenerationEU Fund; and BRIGHT: Best-in-class RapId companion diaGnostics cHip for cancer stratification (nº72328) funded through European Regional Development Fund ( ERDF ), from the European Union through the Northern Regional Operational Programme (NORTE 2020), from Portugal2020. The authors also acknowledge support from the FCT grant (2021.05593.BD) (Adriana Carneiro). Publisher Copyright: © 2023 The Authors
Palavras-chave
Circulating tumour cells Granulocytes Liquid biopsy Microfluidics Analytical Chemistry Environmental Chemistry Biochemistry Spectroscopy SDG 3 - Good Health and Well-being