**4.1.4 Filters**

340 Hepatocellular Carcinoma – Basic Research

Biosciences AB) or Lymphoprep® (Nicomed) to separate the red blood cells from the

OncoQuick® (Greiner) method uses a specific buffer able to isolate the CTC (Balic et al., 2005; Mankin et al., 2002; Paterlini-Brechot & Benali 2007). These methods are usually fast but expensive and found in a context of clinical laboratory used in routine diagnosis (Table 2). Alternative and cheaper methods can be used by preparing in the laboratory the gradient/density buffers. The same tube can contain one, two or three gradient buffers to increase the specificity of the separation between the different cells present in the blood (Gertler et al., 2003; Paterlini-Brechot & Benali 2007; Racila et al., 1998; Zach & Lutz 2006).

The content of different cells has different osmotic pressures. It is possible to expose the samples to buffer(s) that can be hypo-or hyper osmotic to any cell different to the target cells. After the lysis step the mix is centrifuged and the pellets will contain the CTC. Some companies provide a kit with lysis buffers ready to use. After lysis, the next step is the extraction of DNA or RNA (e.g. Red Lysis Buffer from Qiagen or Panomics) or the extracted cells can be purified by immunomagnetic beads enrichment (Aryal et al., 2004; Khan et al., 2000; Wharton et al., 1999). However, lysis buffer can induce the death of a lot of cells including the CTC and it is not appropriate if the sample contains few CTC leading to false

The cytocentrifugation was designed for hypocellular fluids; it spins at lower speeds and has more gradual acceleration and deceleration than normal centrifuges. Some are able to deposit cells directly onto a slide for examination. Cytocentrifugation could be used in research purposes and is also widely used in the routine surgical pathology practice. This method is fast and affordable (Becker et al., 2005; Kallergi et al., 2008; Kollermann et al.,

Methods to identify CTC can after be used (see below). As it occurs with magnetic beads, cytospin increases mortality of the target cells (Table 2). Because enrichment by cytocentrifugation is a critical step, addition of 10 % buffered formaldehyde solution added to the blood sample can preserve morphology of the cells and will certainly preserve nucleic acids integrity (Farina et al., 2004), but the disadvantage of this method is that formaldehyde kills the cells (Table 2). Liquid based cytology (LBC) using a filtration process and computer assisted thin layer deposition of cells has been developed as a replacement for cytocentrifugation and/or smearing, owing to its improved cell recovery capabilities and better cell preservation. In most published series, LBC allows a good interobserver reproducibility. In the urine, processing by the Cytyc ThinprepH 2000 system (Cytyc Corp, Boxborough, Massachusetts, USA) is a method that combines centrifugation and filter transfer methods. The vial containing CTC is placed into a processor (the machine which prepares the smears) together with a glass slide and filter mechanism. The processor immerses the filter assembly into the vial and spins it at a high speed to ensure an even mix of the cells and to break up large cell groups. The fixative is then sucked through a filter membrane which traps the cells but allows fluid through. When an adequate number of

other cells including CTC (Table 2).

**4.1.2 Lysis buffers** 

negative results.

1999; Molnar et al., 2001).

**4.1.3 Cytocentrifugation (cytospin)** 

A non-specific method of enrichment using filters can captured the cells with a certain size. The cells captured on the filter can after be transferred and analyzed on a slide. In this case the samples can come from blood or body fluids (urines, cerebrospinal fluid or ascites). We will describe two kinds of methods using this technology and usually used to isolate CTC (Table 2): the Isolation by Size of Epithelial Tumor Cells (ISET) method and the Micro-Electro-Mechanical System (MEMS).

**The Isolation by Size of Epithelial Tumor Cells** (ISET) method (Metagenex, Paris, France, www.metagenex.fr) separates cells by size with a filter. Cells larger than 10 µm, including tumor cells from carcinomas, are enriched from leukocytes (erythrocytes are lysed, see above) on a filter. Enriched cells are stained on the filter and CTC are precisely counted after cytopathological evaluation. The cells on the filter can be also studied by immunolabelling,

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 343

to grow-up in a specific culture cell medium. A method (EPISPOT) that combines the capacity of CTC to secret specific markers and grow-up in specific cell medium was

To use immune-magnetic detection system the first step is to deplete the whole blood of the red cells (by lysis buffers or density gradient) to obtain the PBMC. After, the magnetic particles coated and surrounded by a specific antibody are added to the PBMC supposing containing the CTC. Labeled cells are then collected by applying a magnetic force while non labeled cells are containing in the supernatant and are discerned. This use of magnetic beads to catch specifically CTC is called "positive selection" (Alix-Panabieres et al., 2008; Paterlini-

Since a large number of leukocytes (potential source of false negative CTC) still remain trapped with the cells, some methods include a "negative selection" of leukocytes (with anti-CD 45 beads for example) followed with a "positive selection" with antibodies specific to epithelial cells (EpCAM, CK) (Allard et al., 2004; Paterlini-Brechot & Benali 2007; Smirnov et al., 2005). The problem of this procedure is that gets ride the majority of leukocytes but still hold in non-malignant epithelial cells and loses tumor cells which do not express epithelial antigens and/or are lysed during the first step (Paterlini-Brechot & Benali 2007;

The methods using antibodies like immune-magnetic methods (MACS system, Deanabeads® Invitrogen, macro-iron beads, ferrofluid(colloidal iron)-based systems) to enrich the sample will induce false-positive extraction (Sergeant et al., 2008). For example, antibodies against cytokeratin (CK) or other epithelial-specific antigens have been reported to bind both specifically and non-specifically to macrophages, plasma cells and nucleated hematopoietic cells precursors. The non-specific binding of the antibodies involves Fc receptor-bearing leukocytes and monocytes or illegitimate expression of epithelial antigens in normal hematopoietic cells. Some of these positive cells are morphologically difficult to distinguish from CTC. Variable numbers of epithelial cells have been found in peripheral blood of subject without malignancy in some physio-pathological conditions like benign epithelial proliferative diseases, inflammation, surgeries and tissue trauma (Allard et al., 2004; Becker et al., 2005; Goeminne et al., 2000; Naume et al., 2004; Naume & Espevik 1991; Paterlini-Brechot & Benali 2007). Moreover, epithelial CTC may lose epithelial markers during dissemination through the process called epithelial-to-mesenchymal transition (EMT). Since the epithelial markers that get lost during EMT may include markers used for CTC measurement, underestimation of the actual CTC number may occur, inducing de facto false negative results (Christiansen & Rajasekaran 2006; Paterlini-Brechot & Benali 2007; Sleijfer et al., 2007; Wang J. Y. et al., 2006; Willipinski-Stapelfeldt et al., 2005). In the case that the method induce false positive, the problem can be diminished avoided using a second marker or a full panel of markers and techniques (see below) to characterize the CTC, like RT-PCR, immunocytochemistry or immunefluorescence, morphology by optical microscopy (Paterlini-Brechot & Benali 2007; Schuler & Dolken 2006; Sleijfer et al., 2007). In another hand, in the case of the false negative results the doubt persist, and only strict follow-up of the patient by repeating the detection of the CTC can potentially eliminate this doubt. No available antibodies are 100% tumor or tissue-specific (El-Serag 2004; Goeminne et al., 2000;

developed (see Table 2).

Zigeuner et al., 2003).

**4.2.1 Magnetic separation** 

Brechot & Benali 2007; Sleijfer et al., 2007).

FISH, TUNEL and molecular analysis. Molecular analysis can be performed specifically to CTC after laser microdissection. The filter can be also mounted between slide and coverslip for routine microscope observation and storage. Although promising, this method is expensive, time consuming and the filters are not easily available (Mejean et al., 2000; Paterlini-Brechot & Benali 2007; Pinzani et al., 2006). Cells should be better characterized using morphological methods that allow both detection and characterization. A second potential main advantage is that CTC could be compared to the primary tumor in order to better understand the mechanism of metastatic process. However, this approach has been rarely performed and neither firm recommendation nor conclusion could be drawn. This method has a high sensibility since one tumoral cell could be detect in 1 ml of blood. The technique also avoids damaging the tumor cell which can be diagnosed using a simple pathologist analysis. However, the pathologist should get used to this technique to avoid a misinterpretation with others types of cells. The use of ISET technology to detect and characterize CTC in HCC has been reported in one study. Vona et al., (Vona et al., 2004) reported that microemboli and isolated CTC could be detected in HCC patient. Presence of CTC was associated with a shorter survival. This work also showed the feasibility of molecular studies of individual circulating cells. Indeed, β-Catenin mutations were searched in samples of 60 single microdissected CTC. β-Catenin mutations were found in only 3 CTC that highlighted the weak impact of these mutations in the initial step of tumor cell invasion. Further studies are needed.

**The Micro-Electro-Mechanical System (MEMS**)-based is a parylene membrane microfilter device for single stage capture and electrolysis of circulating tumor cells in human blood, and the potential of this device to allow genomic analysis. After the CTC are captured in the filter, electrical lysis of cells on membrane filter is applied and the DNA as well as RNA can be extracted and analyzed by PCR or RT-PCR respectively. CTC enrichment is performed by either gradient centrifugation of CTC based on their buoyant density or magnetic separation of epithelial CTC, both of which are laborious procedures with variable efficiency, and CTC identification is typically done by trained pathologists through visual observation of stained cytokeratin-positive epithelial CTC. These processes may take hours, if not days. The Micro-Electro-Mechanical System (MEMS)-based makes the process simpler, faster and better to separate CTC (~90% recovery) from blood cells. Since enrichment will inevitably be accompanied by loss of CTC, irrespective of the exact method, some essays are performed directly in whole blood (Lu Y. et al., 2007). But the disadvantages of this technique are that morphology of the cells is lost, besides markers also and the capacity to count exactly the number of CTC (Table 2).

#### **4.2 The specific enrichment techniques**

The specific enrichment techniques can use specifically protein tumoral markers expressed by the CTC. These methods use antibodies against the protein tumoral markers coupled to steel beads, by applying a magnetic field the cells expressing the marker can be captured. Several immune-magnetic methods (MACS system, Deanabeads® Invitrogen, macro-iron beads, ferrofluid(colloidal iron)-based systems) to enrich the sample have been used successfully (Sergeant et al., 2008). We will describe the methods and their advantages and their disadvantages. Another approach to enrich the sample is to use the properties of CTC to grow-up in a specific culture cell medium. A method (EPISPOT) that combines the capacity of CTC to secret specific markers and grow-up in specific cell medium was developed (see Table 2).
