**2. ctDNA methodological approaches and technical considerations**

Circulating cell-free DNA (cfDNA) was first detected in 1948 by Mendel and Metais in the peripheral blood plasma of healthy and diseased individuals [8]. CfDNA levels can vary between 1 and 10 ng mL<sup>−</sup><sup>1</sup> in plasma and can be affected by physiological conditions such as exercise and acute inflammation [9]. In 1977, Leon et al. found that cfDNA was more elevated in cancer patients compared with healthy subjects, with higher levels correlating with higher burden of disease [10]. In 1989, Stroun et al. discovered that at least part of the plasma DNA in cancer patients originated specifically from cancer cells [11]. In the ensuing decades, knowledge and applications of tumour-derived cfDNA has rapidly evolved due to advances in molecular techniques, and also gave rise to the term, circulating tumour DNA (ctDNA).

**57**

*yield and purity [5].*

**Figure 2.**

*Current Utility and Future Applications of ctDNA in Colorectal Cancer*

clinical relevance, it is prudent to reflect on these biological variables.

Numerous inherent challenges have affected the development of ctDNA preanalytic and analytic methods. These include variable fragmentation, low abundance in plasma or serum volumes, tumour heterogeneity, and low stability as a

To minimise sample degradation and optimise stability, a number of preanalytical steps need to be carefully planned. Although there are currently no standardised methodology guidelines on ctDNA collection, storage and processing,

A variety of methods for detecting and characterising ctDNA have been reported. These can be broadly categorised as targeted and non-targeted

approaches. Differing performance characteristics, strengths and disadvantages may also facilitate complementary roles of these approaches in molecular analysis. **Table 1** lists examples of described methods. Applying any of these approaches in

*Pre-analytical components in ctDNA analysis. (1) Collection of blood samples (usually 5–10 mL) via phlebotomy. Currently, there is no guidance on the comparative impact of the source of blood draw (for example, peripheral venepuncture or intravascular ports) on ctDNA analysis [5]; (2) samples should be collected in tubes containing anticoagulants compatible with polymerase chain reaction methods, such as ethylenediaminetetraacetic acid (EDTA) [9]; (3) centrifugation of blood to separate cells should be performed promptly. The exact optimal time to centrifugation is not known and may depend on storage conditions and the presence of stabilising agents [16]. Current evidence suggests that plasma is preferred to serum samples, as in the latter case, cfDNA released during white blood cell lysis may lead to a dilutional effect [9]. (4) Processed plasma is then generally stored frozen, often in aliquots; (5) CfDNA is extracted using commercially available kits. There are multiple DNA purification strategies and modifications, which may variably impact on DNA* 

A variety of tumour-specific molecular alterations may be identified by ctDNA including mutations, methylation variants, microsatellite alterations, copy number variations and structural changes [12]. Although the exact mechanisms are yet to be elucidated, ctDNA is thought to be released into the blood stream via biological processes such as apoptosis, necrosis, inefficient phagocytosis and active secretion [13, 14]. CtDNA has a short half-life of up to a few hours and accounts for generally only a small fraction of cfDNA, although concentration can vary widely from <0.01 to 90% [12]. The biological and tumoural determinants underlying ctDNA variations both between and within individuals are incompletely understood, but are likely affected by tumour burden, treatment response, circulatory elements, circadian rhythm, cellular turnover and clearance mechanisms [12, 15]. Somatic variants may also be found in healthy individuals, mostly commonly associated with clonal haematopoiesis [5]. Such variability, coupled with the often-low allele frequency of the molecular aberration of interest, demand sensitive and robust detection methods. As we interpret the results of ctDNA studies and consider their

*DOI: http://dx.doi.org/10.5772/intechopen.82316*

**2.1 Pre-analytic considerations**

**2.2 Detection methods**

result of the aforementioned biological factors [16].

the typical workflow is illustrated in **Figure 2**.
