Modern Techniques in Cytopathology. Группа авторов
2. Cell pellet processing: the cell pellet can be processed with or without the addition of a congealing agent.
In general, some of the common steps among the various cell block techniques include specimen concentration, usually by means of centrifugation, supernatant removal, and cell pellet formation (Fig. 1). One of the main differences lies in the method(s) to form the cell pellet to keep the cell block sample intact, so that it can be placed in a cassette for processing and paraffin embedding similar to a histology sample [6].
One common way to maintain the integrity of the pellet and prevent dissolution during processing and embedding is to add a congealing agent (Fig. 2). HistoGelTM (Thermo Scientific Richard-Allan Scientific, Waltham, MA, USA) [7] is frequently used to achieve this goal (Fig. 3). A commercial agarose-based product, HistoGel requires heating from its gel-like state to a liquid form. Per the manufacturer’s instructions, 4–6 drops are then carefully added to the cell pellet; the combination of the two may either be vortexed or mixed together with a swirling motion and then cooled down, allowing the cell and HistoGel mixture to solidify, forming a congealed cell pellet at the bottom of the test tube. The final pellet will assume the shape of the tube in which it is solidified. In the case of a centrifuge tube, this can result in a cone-shaped cell pellet and an uneven distribution of cells, which may in turn lead to uncertainty about the optimal orientation for the pellet during embedding. A marker, such as suture material, may be added to the mixture to indicate the location of the cells within the block and how deep to trim the block in histology [8]. Such markers are particularly useful when the cell block is paucicellular, and when the cellular portion of the sample is clear or light-color camouflaged in HistoGel and paraffin.
Fig. 3. HistoGel method: following centrifugation and decanting steps, liquefied Histo-Gel (purple) is added and then allowed to solidify with cells before placing the pellet into a cassette.
Fig. 4. Plasma-thrombin method: following centrifugation and decanting steps, an aliquot of plasma (yellow) is added to the pellet; this is vortexed and then thrombin (green) is added. After clot formation, the combination is placed in a cassette.
Another frequently used cell pellet-congealing method employs plasma-thrombin (Fig. 4). Typically, this method utilizes expired plasma from a blood bank as well as commercial thrombin. Commercial plasma is also available for centers without access to blood banks at significantly higher cost. The plasma and thrombin are added to the cell pellet and allowed to clot and form a coagulum. When expired plasma from the blood bank is used, the clotting factors will vary and the plasma may not be able to form a coagulum consistently [9]. Additionally, there are also different types of thrombin, with some requiring reconstitution and activation [10]. Important limitations of the plasma-thrombin method are the collection, holding media and potential for DNA contamination, especially with development of increasingly sensitive molecular testing platforms [11]. Placing a specimen intended for a plasma-thrombin cell block in formalin or alcohol can compromise the pellet since both of these are anticoagulants and will block the clotting cascade. For FNA biopsies, saline or Roswell Park Memorial Institute (RPMI) medium, which is much more expensive, are alternatives, but neither of these is a fixative [12], and specimen integrity may be compromised in cases of extended interval between specimen collection and processing.
There are several other traditional cell block methods. One method that is simple in approach involves centrifuging the sample and simply placing the pellet in a mesh bag or wrapping it in filter paper without adding any coagulating reagent [13]. CytoRich Red Collection Fluid (Thermo Scientific Richard-Allan Scientific), an alcohol-based fixative, may be used with HistoGel or plasma thrombin to create a dense and compact block. This is achieved by placing the combination of cell pellet and congealing reagents into filter paper or a mesh bag and squeezing out the excess fluid [14]. An alternative approach is to pour the sample through filter paper lining a funnel with the intention of capturing all cells and tissue fragments (Fig. 5). After filtering, the paper is folded and placed in a cassette and processed as a histology sample – the entire paper is embedded in paraffin and sectioned when preparing slides. With this method, formalin is typically the rinsing agent, although any liquid media can be used. There may be loss of some of the free-floating single cells with this method. A variation on this technique is to add some HistoGel to the filter paper after filtering to help trap the smaller tissue particles.
Fig. 5. Filter paper is folded into a cone shape, and the specimen is poured through it leaving behind the cells in the filter paper. The filter paper containing the specimen is folded and submitted to histology.
Fig. 6. The entire specimen is expelled onto a glass slide, where the sample is allowed to clot and then placed into media.
The clot method, which is used for FNA samples, involves expressing the aspirate material sample from the needle with a syringe or with a stylet, directly onto filter paper, a glass slide or other surface, where it is then allowed to clot (Fig. 6). The clot is then submitted in a cassette for processing in histology without the addition of a congealing agent [15]. The Shandon Cytoblock (Thermo Scientific Richard-Allan Scientific) concentrates cells in the Cytoblock cassette. A sample is added through a Cytofunnel connected to a Cytoblock cassette, and it undergoes centrifugation with cytospin. Upon completion, the Cytoblock cassette containing the sample is placed in a tissue processor, like other tissue samples, and the cell pellet is released from its board insert in the Cytoblock for paraffin embedding [16]. A salvage “cytoscrape” cell block technique, used in the absence of material available/collected in media, involves removing the coverslip from a stained smear, scraping off the cells, and centrifuging with agarose to form a pellet [17].
Other traditional cell block methods include sedimentation, agar, albumin, natural clot formation, zinc formalin, glucomannan [18], and a combination of three agents (ethyl alcohol, formalin, and glacial acetic acid) [4], among others. The collodion bag method [19]