Table 2.

Common preanalytical issues and recommendations

Preanalytical issue	Challenges	Recommendations
Isolation of disease-specific analytes from biofluids (CTCs, ctDNA, EVs)	lack of standardized protocols for processing of biofluids poor consistency among studies of reported kits employed for analyte isolation	need to establish standard sample handling and collection protocols use of extensively tested and validated commercially available kits for optimal analyte isolation
Study population selection	convenience sampling commonly used to validate proposed mutation detection assays suboptimal control population	multi-institutional collaborations to design a comprehensive patient population for improved generalizability of reported results selection of appropriate controls (healthy, benign disease of the same organ)
Confounding biological and environmental variables	influence of pre-sampling factors on quality of isolated analytes (circadian rhythm, fasting, metabolic disorders, hypertension, pregnancy, lactation)	inclusion of recommendations in sample collection protocols to improve rigor and reproducibility
Long-term sample storage conditions and biobanking	inconsistent data on decay rates reported with the use of different storage methods freeze-thaw cycles and thawing procedure can influence target nucleic acid concentration and integrity	need to determine optimal storage conditions (time, temperature) for cell-free matrices and extracted nucleic acid evaluation of appropriate thawing procedure (duration, i.e., fast vs. slow, temperature) and sample storage volumes
Inclusion of study design specific controls	limited understanding of biological variation in individual samples as well as patients’ own biofluids over the course of disease	use of internal synthetic, known standards, to better account for biological variation in the biofluids and technical variation in reported techniques