**4.1.2 Lysis buffers**

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 negative results.

#### **4.1.3 Cytocentrifugation (cytospin)**

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., 1999; Molnar et al., 2001).

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

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 technologists' screening time (Table 2).

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).
