Modern Techniques in Cytopathology. Группа авторов

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 cellular ones, as the latter are likely to produce diagnostic cell blocks even with less than optimal methods.

      Cell Blocks: Fixation and Ancillary Studies

      Besides the use of various processing techniques, cell block samples are also collected in different media. There is no standard for what media are used to initially collect cell block samples, yet this may affect ancillary test results performed on cell block sections. For FNA samples, media that have been used over the years for rinsing the needle aspirate include saline, RPMI, Hank’s solution, Bouin solution, formalin, and alcohol, amongst others. The rinsing media also serves as holding or transport media in most cases. Saline or RPMI are used when there is a need for flexibility with how the sample is processed (e.g., to perform flow cytometry or submit for microbiology cultures). This flexibility comes at a cost, as neither saline nor RPMI can be perfectly isotonic with the cells in an FNA sample, and when not isotonic, the saline may cause cell membrane lysis with cell blocks showing stripped nuclei and cytoplasmic fragments [12].

Fig. 7. Excess congealing agent (HistoGel). a Addition of excess congealing agent, represented by amorphous pink material in the background, dilutes the specimen. b Using saline may alter the cytomorphology.

Formalin and alcohol are also employed commonly in cytology. Alcohol, as a rinsing and transport medium, causes less degradation of DNA and RNA than formalin and may be better suited as a fixative for molecular testing [30, 31]. However, formalin is much more commonly used and remains the gold standard fixative used for validation of most ancillary studies, including both IHC and molecular assays. Unlike alcohol, formalin intercalates in DNA strands and may cause random alterations in nucleotide bases, leading to false positivity, especially when using sensitive assays [31]. Formalin is also known to cause DNA fragmentation,