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, which typically does not impact analyses of short DNA segments but could result in unsuccessful molecular analyses with greater lengths of DNA [31]. Molecular analyses on specimens placed in acidic solutions (e.g., Bouin solution, decalcifying solution) and heavy metal fixatives (e.g., B5, Zenker, and acid zinc formalin) should not be performed; most of these agents have fallen out of favor for use as fixatives in cytology for rinsing, holding, or transport media.
Since most IHC are developed using formalin-fixed paraffin-embedded (FFPE) surgical pathology tissue sections, deviation from this standard may impact IHC staining of cytology cell block sections [32]. Therefore, when alcohol or other non-formalin fixative is used, validation is necessary to confirm IHC results [30]. CACBS uses CytolytTM, a proprietary alcohol-based fixation medium, and formalin is contraindicated with this method’s processor [22]. Results of IHC-processed cell blocks using alcohol and CACBS are variable with some stains demonstrating correlation with FFPE samples and others showing inconsistencies (e.g., lack of staining and/or weaker intensity) [24, 28, 33]. Examples include inconsistent staining for cytokeratin (CK) 7, CK20, WT-1, calretinin, thyroid transcription factor-1 (TTF-1), estrogen receptor, and progesterone receptor [33], among others.
Possible solutions to circumvent this have been described, such as pre-fixation of the sample with formalin followed by a CytoLyt wash prior to placing in PreservCyt [29], or post-fixation of a CACBS-produced slide in formalin [33]. Some of these techniques have shown inconsistencies when studied [30], and further investigation is thus necessary to meet guidelines set forth by the College of American Pathologists (CAP) requiring a minimum of 90% concordance between an existing and new test [34]. The impact of different fixatives is not limited to IHC but may also impact cytochemical or “special stains” such as PAS-D or mucicarmine [24]. Overall, studies indicate that regardless of the fixative and cell block preparation method used, appropriate validation is a crucial component to ensure accurate results.
Cell Blocks: Morphology
The effect on morphology by fixative (alcohol and formalin) and different cell block preparations has been examined. Similar to outcomes reported for cellularity and ancillary studies, these results also vary. In general, alcohol fixation is associated with shrinkage artifacts and better nuclear detail [12]. Some investigators have detected no significant difference between traditional methodology (e.g., HistoGel, plasma-thrombin) and CACBS [21, 28, 30], whilst others have noted a statistically significant improvement in the appearance of the cytoplasm, nucleoli, and chromatin in CACBS-prepared cell blocks [24, 29], attributable to its alcohol-based fixation [33].
Cell Blocks: Evolution, Emerging, and Modern Techniques
The cytology community continues to investigate and explore solutions to provide quality cell blocks that meet the demands of personalized medicine and that overcome the limitations of traditional cell block techniques and CACBS. This has involved revisiting techniques introduced decades ago (i.e., collodion bag), modifying existing ones (i.e., Cell-Gel, AFFECT), and investigating new ones (i.e., XCellent; Fig. 8). The ideal method would generate cell blocks that have high cellularity from capturing all tissue fragments and single cells with minimal cell loss during processing, integrate well with existing methods and workflows, utilize fixation of existing validated methods, not be labor intensive [10], require minimal skill, and be economical. A combination of these features would facilitate standardization across laboratories.
To achieve these milestones, the limitations of existing methods need to be addressed. Low cellularity, one of the most significant deficiencies [7], is typically due to one or more factors – both operator and cytology dependent. In terms of cytology procedures, the inability to capture all of the cells, the lack of a tightly cohesive pellet, and/or dilution by a congealing agent are often reasons for low cellularity in the cell block.
The collodion bag technique, first described in 1993 by Bedrossian et al. [19], has been updated and given the moniker “the Method of Mah” after
