**4. Enrichment of the sample**

CTC are usually detected in the peripheral circulation, but we can find CTC in other body fluids like the cerebrospinal fluids or the urines. The limitations to discover the CTC in these fluids are the same than in the blood circulation. However, it is possible to extract a relatively big amount of blood without harming the patient and much easier.

We will focus on the methods of CTC detection in the blood. As we describe above, CTC in the peripheral circulation occur at an estimated number of one CTC per 105-7 peripheral blood mononuclear cell or PBMC. Because of the scarcity of the target cells, it is necessary to concentrate the sample. Since enrichment will inevitably be accompanied by some loss of CTC, irrespective of the method, some essays are performed directly in whole blood (Lu Y. et al., 2007).

Two different groups of techniques can be used to enrich samples, the non-specific and the specific enrichment techniques. The non-specific enrichment techniques use physicochemical CTC properties (size, density, etc). The specific enrichment technique use markers expressed by the CTC. The advantages of the non-specific and specific enrichment techniques are summarized in the Table 2 and described in the following paragraphs.

Around 28 years ago, microRNA (miRNA) was discovered and showed the regulation of genes (Lee et al., 1993; Reinhart et al., 2000) such as oncogenes or tumor suppressor genes at the level of the mRNA. More than 35 studies focused on the identification of miRNA or a group of miRNAs to be used as marker of early diagnosis or metastasis. miR-122/-122a, miR-221/222, miR-145, miR-146a, miR-26 (NFB pathway), miR-199a-3p (mTOR pathway) and miR-26 (MYC pathway) were strongly linked to the development and metastasis of the HCC. Also a group of miRNAs were used to identify and classify HCC (Hoshida et al., 2010; Ji & Wang 2009; Kerr et al., 2011; Kojima et al., 2011; Kong G. et al., 2011; Sato et al., 2011),

In Conclusion, we can observe that not too many specific HCC markers are available and useful for the detection of the CTC. This is certainly due to the heterogeneity of the hepatocellular carcinoma. The most important marker used in clinical routine is the detection of serum AFP mRNA expression (Table 1). But this marker is not expressed in all HCC and by consequence in all CTC leading false negative results. Some propose to combine the research of more than one marker to increase the specificity and the sensitivity of CTC detection method. One of promising marker is Cancer-Testis Antigens but more studies need to be done to select one or more CTA combined (or not) with the detection of the AFP mRNA expression. As we notice previously, CTC are very rare in peripheral blood. We saw also that real-time polymerase chain reaction is a method that in addition to be specific by the nature of the primers used, it can amplify the signal by increasing the number of copies of mRNA originally presents in the sample. But before using RT-PCR, it's necessary to concentrate the number of CTC from the peripheral blood in a smaller volume.

CTC are usually detected in the peripheral circulation, but we can find CTC in other body fluids like the cerebrospinal fluids or the urines. The limitations to discover the CTC in these fluids are the same than in the blood circulation. However, it is possible to extract a

We will focus on the methods of CTC detection in the blood. As we describe above, CTC in the peripheral circulation occur at an estimated number of one CTC per 105-7 peripheral blood mononuclear cell or PBMC. Because of the scarcity of the target cells, it is necessary to concentrate the sample. Since enrichment will inevitably be accompanied by some loss of CTC, irrespective of the method, some essays are performed directly in whole blood

Two different groups of techniques can be used to enrich samples, the non-specific and the specific enrichment techniques. The non-specific enrichment techniques use physicochemical CTC properties (size, density, etc). The specific enrichment technique use markers expressed by the CTC. The advantages of the non-specific and specific enrichment techniques are summarized in the Table 2 and described in the following

relatively big amount of blood without harming the patient and much easier.

but non of them were used as a CTC marker and tested during a clinical trial.

**3.3.3 MicroRNA markers: A new hope** 

The next chapter will describe these methods.

**4. Enrichment of the sample** 

(Lu Y. et al., 2007).

paragraphs.

**3.4 Conclusion** 


Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 339

The tumor cells, epithelial cells, platelets and low density leukocytes from leukocytes and erythrocytes can be separated by the propriety of their particular density (Table 2). Briefly, each cell type has their own density and the assumption of the methods is to put the cells in a buffer with a specific kind of density that usually corresponds to the density of the cells (CTC) that we want to isolate. Density gradient centrifugation is the preferred method to purify cells, subcellular organelles and macromolecules. Density gradients can be generated by placing layer after layer of gradient media such as sucrose in a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous or continuous mode. The cell fraction to be separated is placed on top of the layer and centrifuged. Density gradient separation can be classified into two categories: 1). Rate-zonal (size) separation. 2).

Rate-zonal separation takes advantage of particle size and mass instead of particle density for sedimentation. The examples of common applications include separation of cells, cellular organelles such as endosomes or separation of proteins, such as antibodies (Rickwood D;




In isopycnic separation, a particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. Once this quasi-equilibrium is reached, the length of centrifugation does not have any influence on the migration of the particle. A common example for this method is separation of nucleic acids in a CsCl gradient. A variety of gradient media can be used for isopycnic separations (Rickwood D; Grahm J. M. 2001).



In the context of the CTC enrichment by centrifugation the isopycnic separation is the method usually used. The cells that have a density higher than the density of the buffer will stay in the bottom of the tube. If the density of the cells is lower than the buffer, they will remain on the top of the liquid, forming a ring. On the contrary, if the density of the cells is the same than the buffer, the cells will form a ring in the middle of the tube. A well known example of the method is the commercial buffer FICOLL™ tube (Amersham

**4.1 The non-specific enrichment techniques** 

Criteria for successful rate-zonal centrifugation are:

**4.1.1 Density gradient(s) centrifugation** 

Isopycnic (density) separation.

Grahm J. M. 2001).

the gradient.

portion of the gradient.

Criteria for successful isopycnic separation:



Table 2. Summary of advantages and disadvantages of the methods of CTC enrichment. CTC, circulating tumor cell; CTC-Chip, circulating tumor cell chip; EPIPSPOT, epithelial immunospot; FACS, Fluorescence-activated cell sorting = flow cytometric; FAST, fiber-optic array scanning technology; ICH, immunocytochemistry; ISET, isolation by size of epithelial tumor cells; MACS, magnetic cell sorting; MEMS, micro-electro-mechanical system.
