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REVIEW

Pre-analytical considerations for microRNA quantification in childhood leukemia research

Ioannis Kyriakidis1* ,  Iordanis Pelagiadis1 ,  Eftichia Stiakaki1
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1 Department of Pediatric Hematology-Oncology and Autologous Hematopoietic Stem Cell Transplantation Unit, University Hospital of Heraklion and Laboratory of Blood Diseases and Childhood Cancer Biology, School of Medicine, University of Crete, Heraklion 71003, Greece
JBM, e99010062 https://doi.org/10.14440/jbm.2025.0001
Submitted: 1 January 2025 | Revised: 26 March 2025 | Accepted: 15 April 2025 | Published: 27 May 2025
© 2025 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

Background: MicroRNAs (miRNAs) have gained significant attention as potential biomarkers in childhood leukemia, offering insights into diagnosis, prognosis, and therapeutic targeting. However, the clinical translation of miRNA biomarkers faces several challenges, including inconsistencies in results caused by methodological differences, sample processing variability, and a lack of standardized normalization techniques. In addition, miRNA profiles are highly cell-specific, with unique signatures for different blood cell types and leukemic blasts, reflecting their distinct biological roles and disease states. Pre-analytical factors are critical in ensuring the accuracy and reproducibility of miRNA quantification. The selectively enriched and highly stable exosomal miRNAs have shown great promise for studying intercellular communication and disease-specific miRNAs. The selection of the analytical matrix should align with the specific objectives of the research or diagnostic application. Addressing technical challenges and recording potential confounding variables (e.g., age, gender, ethnicity, body mass index, menstrual cycle, fasting, circadian rhythm, comorbidities, medications, smoking, and physical activity) are essential to enhancing the reproducibility and reliability of miRNA biomarkers. Objective: This review aims to highlight the challenges facing miRNA quantification in childhood leukemias, scrutinizing all relevant pre-analytical issues, including the preferred specimen source, leukemic blast type and count, the selection of suitable blood components, the impact of time and freeze–thaw cycles, collection, processing, and storage variables. Conclusion: Further research is needed to standardize methodologies and expand our understanding of miRNA interaction networks, ultimately advancing the application of miRNAs in childhood leukemia research.

Keywords
MicroRNAs
Pre-analytical phase
Biomarker
Leukemia
Childhood
Adolescence
Exosomes
Confounding factors
Funding
This work was supported by the Special Account for Research Funds of the University of Crete (grant number: 11515).
Conflict of interest
The authors declare they have no competing interests.
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