Table 1.

Preanalitic variable in liquid biopsy

Variables	Pitfalls	Recommendation
Patient condition	- concentrations of cfDNA increase between physio-pathological conditions: autoimmune diseases, trauma, strenuous exercise, pregnancy - in most early-stage cancers, the amount of cfDNA is very low, similar to healthy subjects	- LB sensibility is higher in patients with high tumor burden
Type of cancer	different tumor types do not release the same amount of ctDNA	- LB sensibility is higher in patients with metastatic cancers of the pancreas, bladder, colon, stomach, breast, liver, esophagus, head and neck and melanoma
Type of blood collection tubes used	risk of WBCs lysis, leading again to ctDNA contamination with wild-type background DNA	- K2/K3EDTA-containing tubes require a short time interval (< 6 h) between blood drawing and sample processing - Specialized blood collection tubes containing a preservative agent maintain stable cfDNA levels for 7 days if stored at RT
Blood processing protocol used	Reduction of cfDNA yield	Double centrifugation step: the first at 1,600 xg, the second at 16,000 xg, 10 min each at 4° C
Plasma storage	Long periods of plasma storage may cause a decreased cfDNA yield	plasma storage for 2 weeks at -20 °C or 4 weeks at -80 °C has no effect on cfDNA extraction
ctDNA storage	Long periods of ctDNA storage may cause DNA fragmentation	Storage ctDNA extracts at -20 °C or preferably at -80 °C, avoid more than three freeze–thaw cyckes
Quality assessment method	Potential false positives are due to clonal hematopoiesis	Assays should incorporate sequencing of leukocytes in addition to plasma DNA