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] is described in more detail below. In a direct comparison of the three most common techniques (plasma-thrombin with saline rinse, HistoGel, and collodion bag), Balassanian et al. [20] were able to demonstrate superior preservation and cellularity using the collodion bag technique. Nevertheless, individual laboratories have adapted different techniques and combinations of these techniques for preparing cell blocks depending on historical precedent, types of samples handled, and familiarity with each method. Overall, there is no universal standard operating procedure for processing cell blocks.
Cell Blocks: Cellient Automated Cell Block System
The Cellient Automated Cell Block System (CACBS; Hologic, Marlborough, MA, USA), introduced in 2006 and built on existing ThinPrep (Hologic) liquid-based cytology, offers an alternative cell block process to overcome some of the shortcomings of traditional methods. Unlike other methods, the CACBS is not as operator dependent, thus providing greater consistency, uses vacuum-assisted filtration to capture all available cells, and employs paraffin as a medium to contain cells. With its many advantages, including automation and standardization, however, come significant associated capital, consumables, and operating costs relative to some of the other traditional methods (e.g., plasma-thrombin, HistoGel, and collodion bags) [21].
CACBS, consisting of two main components – the processor and the finishing station – assumes that some tasks are performed by technicians in the cytology and histology laboratories and yields a paraffin-embedded tissue block, rather than simply a cell pellet, as a final product. This is produced in a significantly shorter overall processing time: approximately 45 min compared to more than 2–8 h for other traditional cell blocks. CACBS accommodates preparing only one block at a time and does not allow for batch processing, so a high-volume laboratory may require multiple instruments.
There are preparatory steps that must take place before the specimen can be processed using CACBS. First, the sample is centrifuged and the supernatant is removed, similar to the traditional methods. An aliquot of the sample is then placed into PreservCyt (Hologic), an alcohol-based preservative. The processor automatically suctions the cells from the PreservCyt vial using vacuum assistance into a CACBS-specific tissue cassette with a detachable filter assembly [22]. Upon completion of the cell collection, the CACBS performs the steps of a tissue processor (e.g., dehydration, clearing, and paraffin infusion). Any tissue fragments in a sample must be manually placed into the cassette using a pipette or forceps by the operator rather than placing them into the processor.
Following paraffin infusion, the automated process cools and hardens the paraffin, after which the filter assembly is removed, leaving behind a circular cell-paraffin pellet attached to the outer surface of the cassette. Final steps occur in the finishing station, in which the cassette is placed into a paraffin-containing metal mold – one similar to that routinely used for embedding in histology laboratories. The CACBS indicates when the cassette is ready to be removed from the embedding mold for slide preparation.
Cell Blocks: Cellularity
A survey across various cell block-processing methods demonstrated that 44% of respondents were either unsatisfied or sometimes satisfied with the quality of cell blocks, with low cellularity cited as the main reason for disapproval [7]. In a comparison of two common methods, plasma-thrombin and HistoGel, respondents in a survey reported greater satisfaction with use of the former, although the difference was not statistically significant.
Low cellularity may be secondary to multiple factors. Some of the reasons may reflect operator skill, nature of the specimen (e.g., spindle cell, sclerotic, or necrotic samples) or scant available cells/volume of available specimen (e.g., minimal exfoliative fluid with very few cells), while others may reflect cytology-related variables. For instance, low cellularity in a cell block may result from poor triage due to smearing most of the specimen on a slide rather than allocating it for a cell block [23]. Additionally, technical expertise and familiarity with processing cell blocks using one of the traditional methods may be a factor. Knowing the optimal amount of congealing agent to add to the cell pellet is significant; an excess will cause specimen dilution and an insufficient amount will result in suboptimal cohesion and cell loss (Fig. 7).
Whether CACBS cell blocks increase cellularity has not been established. When comparing CACBS with agar, Kruger et al. [24] described a significant improvement in cellularity with the former. This was attributed to the ability of CACBS to confine the cells to a defined area and eliminate dilution with a congealing agent. This is best exemplified in low-cellularity specimens, such as FNA biopsies of the breast [25], vitreous [26], and thyroid [27]. In a study of thyroid FNAs, the addition of a CACBS block to a ThinPrep slide resulted in a change from non-diagnostic to diagnostic in 31% of cases [27]. Meanwhile, others have noted no appreciable improvement in cellularity between traditional-type cell blocks and CACBS [28–30]. CACBS may prove more valuable for very scant specimens rather than
