who refined and modernized it [12]. Collodion denotes a syrupy solution. This technique addresses some of the cytology-related shortcomings of more commonly used traditional methods and is particularly well suited for FNA biopsies. In a direct comparison of FNA cell blocks made from saline needle rinses with plasma-thrombin clots, formalin rinses with HistoGel coagulum, and formalin rinses with collodion bags, the collodion bag technique was found to be superior for cell preservation and cellularity [20] (Fig. 9). Rather than using a coagulum, the collodion method traps both free floating cells and tissue fragments in a centrifuged test tube lined by a polymer bag, which is removed, tied off, and submitted in a cassette for routine histology processing.
Briefly, a glass conical test tube is filled with collodion, a commercially available liquid polymer (Cardinal Health Inc., Dublin, OH, USA; Fig. 10). The collodion is then poured out of the test tube, leaving behind a thin lining along the walls of the tube, which is then inverted and allowed to dry completely under a laminar flow hood. This process forms a membrane lining inside the tube. Test tubes can be prepared in large batches and stored for up to a month.
The FNA needle is rinsed into a 10-mL vial of formalin, and the entire sample is poured into the test tube lined with collodion. The test tube is centrifuged to form a pellet. The centrifugal force and osmosis pushes some of the liquid formalin through the collodion membrane lining the tube, allowing it to separate easily from the tube, while trapping the cellular particles inside the bag. The membrane bag is then gently separated from the test tube wall, which forms a collodion membrane bag with the pellet at the bottom of the conical test tube. The bag is gently removed from the test tube. A cotton string is tied around the bag, thereby trapping the pellet at the base of the bag, which is then cut just above the string allowing the remaining supernatant fluid to drain. The bag containing the pellet is placed in a tissue cassette and processed as per routine histology. Working with collodion, an ether-based solvent, requires working in a laminar flow hood and storage in a flameproof cabinet, as the evaporated peroxidases formed from the solvent are flammable [20].
Fig. 8. Cell block preparation methods: several cell block-processing methods, including traditional and newer ones, and those that do and do not use congealing agents are outlined.
Cell-Gel, a recent modification of the HistoGel method, is another economical solution with a decreased reported failure rate [9] when compared to traditional HistoGel cell blocks. Briefly, after a sample is mixed with a hemolytic agent to lyse red blood cells, it is centrifuged, and the supernatant then discarded. The remaining concentrate is transferred into an appropriate-sized disposable mold used during tissue embedding in histology. HistoGel is subsequently added and the assembly is cooled until it has solidified. The solidified pellet is removed from the mold and positioned in a cassette lined by foam, while maintaining its original orientation in the mold, and the Cell-Gel-containing cassette is placed in the tissue processor. The boundaries and flat bottom of the mold allow the cells to distribute evenly in a restricted area along the bottom. Also, preserving the orientation at the time of embedding encourages sectioning of the cellular side first; however, caution has to be exercised to avoid inadvertently trimming off too much tissue from the block. Because a hemolytic agent (e.g., CytoRich Red), which contains alcohols, is used instead of formalin, validation to ensure compatibility with IHC has to be performed.
Fig. 9. No congealing agent (collodion bag). a Without a congealing agent, there is no specimen dilution allowing all small cell particles to also be captured within the collodion bag. b Formalin fixation preserves cellular detail.
Fig. 10. Collodion bag method: a test tube is filled with collodion (green). This is then poured out, leaving behind a thin coating. The specimen is poured into the collodionlined tube and centrifuged. Post-centrifugation, the collodion bag is removed and tied with a string. The excess bag is cut, and the portion containing the cells is placed into a cassette for processing in histology.
Another contemporary technique known as “a formalin-fixed paraffin-embedded cytology cell block technique” (AFFECT) [35] uses aspects of the Shandon Cytoblock. In a direct comparison with the agarose cell block method, AFFECT demonstrated improved cellularity and morphology. The samples for this method are collected in saline and centrifuged. The supernatant is removed, and the concentrate is fixed in approximately 20 mL of formalin for 1–2 h. This is followed by another centrifugation and decanting step; the specimen is vortexed and placed into a cytospin centrifuge. Unique to AFFECT is an absorbent foam receptacle (Infinicel; Procter & Gamble Co., Cincinnati, OH, USA). The foam and tissue paper are placed in between the funnel and metal clip, which are standard components of the cytospin centrifuge. After centrifugation, the foam is wrapped in the accompanying tissue paper, placed in a tissue processor, and embedded such that the open-exposed surface of the foam receptacle lies at the base of the mold. With AFFECT, cell blocks are typically made from specimens that would not have been attempted using traditional agarose. AFFECT has also resulted in superior cytoplasmic and nuclear detail relative to the agarose method; it is likely that the heated agarose negatively impacts cytomorphology [35].
XCellent, a device in development, increased the cellular yield and enhanced the cytomorphology of cell block samples [36], in a direct comparison to HistoGel. Briefly, the specimen is placed in a tube with a detachable base. After standard centrifugation, the base containing the sample is removed, capped, and laid in a cassette for tissue processing. The entire base is embedded in paraffin and sectioned. This method is fixative agnostic and less skill dependent than traditional methods. Unlike the above methods, XCellent is not yet commercially available.
Cell Blocks: Application to Modern and Alternative Techniques
Cell blocks have several advantages, some described more extensively in the literature and others gaining recognition. For instance, cell blocks allow cytology samples to be utilized diagnostically in ways that have traditionally been reserved for surgical pathology specimens. Digital pathology is one such rapidly growing field. Whole-slide imaging (WSI) of surgical pathology slides, which have 2-dimensions in the x and y planes, provide high-resolution images from an entire scanned slide. The Food and Drug Administration (FDA) approved the marketing of a WSI system, Philips IntelliSite Pathology Solutions (PIPS), for reviewing and interpreting FFPE surgical pathology slides for diagnostic use. Cytology specimens were specifically excluded from this approval. Cytology LBC preparations and smears, unlike surgical pathology specimens, have 3 dimensions in the x, y, and z planes. Thus, with conventional light microscopy subtle focus adjustments of the stage are required to bring cells located in different planes into focus. The added z-axis of WSI scanning requires greater slide scanning time and storage of larger files [37], and this limitation prevents the routine use of most commercial WSI systems for cytology practice. However, cell block slides (akin to “tissue sections”) by converging cytology and histology, are 2-dimensional and thus could offer
