**4.1 The non-specific enrichment techniques**

## **4.1.1 Density gradient(s) centrifugation**

338 Hepatocellular Carcinoma – Basic Research

**Specific** 

1. Subjective analyses for CTC identification, 2. Time-consuming screening of tumor cells,

(Alix-

(Alix-

Panabieres et al., 2005; Alix-Panabieres et al., 2008; Alix-Panabieres et al., 2007; Czerkinsky et al., 1983)

(Lobodasch et al., 2007; Mankin et al.,

Pachmann et al., 2005)

(Curry et al., 2004; Hsieh et al., 2006; Lu Y. et al., 2007; Pinzani et al.,

Panabieres et al., 2008; Nagrath et al.,

2002;

2006)

(Alix-

2007)

Panabieres et al., 2005; Alix-Panabieres et al., 2008; Alix-Panabieres et al., 2007; Czerkinsky et al., 1983)

marker(s) and antibody

actively secreted, shed, or

2. No identification and isolation of secreting cell

1. Need the apparatus,

1. Fluorescent dyeconjugated antibodies, 2. Specificity depend of

the antibodies, 3.Very expensive.

1. Detect only cytokeratin+-CTC, 2. Need to control precisely laminar flow

conditions, 3. Expensive.

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.

3. Need specific

1. Protein must be

available, 4. Expensive.

released,

possible.

2. High cost.

**ImmunoMagn etic Beads**  (MACS system, CellSearch)

1. Specific

FISH...etc,

on CTC,

**EPISPOT** 1. High sensitivity,

**FACS** 1. High sensitivity,

**FAST** 1. High sensitivity,

**CTC-Chip** 1. High sensitivity,

suspended,

2. Morphological analysis of CTC, Cytopathology, Cytological staining, ICH,

3. Multiple labelling of antigens

4. Direct quantification of CTC.

2. Detection of viable CTC, 3. Detection of secreted proteins.

2. Technique for counting, examining, and sorting microscopic particles (CTC)

chemical characteristics.

2. Can detect rare events.

2. Detection of viable CTC.

3. Simultaneous multiparametric analyses of the physical and/or

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). Isopycnic (density) separation.

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; Grahm J. M. 2001).

Criteria for successful rate-zonal centrifugation are:


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).

Criteria for successful isopycnic separation:


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

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 341

cells have been deposited on the filter, the processor detects a drop in the suction pressure and stops drawing fluid through the filter. It then applies the filter to a specially prepared glass slide and transfers the cells across. The slide is deposited into a vial of fixative (paraffin 10%) from which it is subsequently taken out and stained. It results in increased cellularity and a pronounced reduction of debris, red blood cells (RBC), and crystals (Papillo & Lapen

There are several advantages to this system. One is that it produces a thin layer of cells which is easier to evaluate than a thick smear. The morphology of the cells is also better. In addition, the entire cell sample is captured in the fixative vial which leads to a more representative smear being prepared. One of the most important advantages of this test is that the material that is left over after a smear has been prepared can be used for adjunctive testing. A further advantage is that the smears may be initially subjected to image analysis. Computer software "reads" the smears and registers the co-ordinates of the fields with what it regards as the most abnormal cells. On review the system directs the cytotechnologists to these fields where they are evaluated. This can cut down on

There are also some disadvantages, which include increased manpower needed to prepare the smears, and the dependence of smear preparation on the instrument. This technique cannot be used directly from blood samples. The red blood cells need to be eliminated by FICOLL® method for example and after the sample can be processed by this technique (Table 2). This method is usually used for urine or ascites samples. However, optimization of cell capture and fixation can be achieved by methods other than Cytyc Thinprep LBC, particularly while using meticulous modern cytocentrifugation methods in the study of hypocellular fluids like in urine for the bladder cancer (Piaton et al., 2004; Wright & Halford 2001). In their study, Piaton et al., conclude that Cytyc Thinprep LBC and modern cytocentrifugation techniques are appropriate methods for cytology based molecular studies. From an economical point of view (standard cytocentrifugation are around \$ 538 compared to Cytyc ThinprepH \$ 1,278 ), and taking into account the value of a meticulous technique, cytocentrifugation with disposable sample chambers remains the quality

standard for current treatment of urinary samples for example (Piaton et al., 2004).

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-

**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,

1994; Piaton et al., 2004; Wright & Halford 2001).

technologists' screening time (Table 2).

Electro-Mechanical System (MEMS).

**4.1.4 Filters** 

Biosciences AB) or Lymphoprep® (Nicomed) to separate the red blood cells from the other cells including CTC (Table 2).

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).
