Modern Techniques in Cytopathology. Группа авторов

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 an alternative for WSI of cytology material. Indeed, Tawfik et al. [37] have described the use of WSI on cell blocks prepared from residual gynecological Pap samples. Based on 1,110 Pap test slides prepared and analyzed using WSI, the sensitivity reported by these authors was comparable to standard LBC-prepared slides and highly specific for identifying low-grade intraepithelial lesions and high-grade squamous intraepithelial lesions. Given that cell blocks are somewhat comparable to surgical pathology specimens, they may have potential for WSI diagnostic use.

Tissue microarrays (TMAs), a high-throughput, faster, and more economical method for comparative IHC or molecular analyses, are generally derived from surgical pathology specimens. They are constructed by removing cylindrical cores of tissue from separate “donor” FFPE blocks, which are then assembled together in a single “recipient” paraffin block. Since cytology microarrays [38] have been constructed largely from cells, they may not afford the same advantages associated with TMAs derived from FFPE blocks. With adequately cellular cell blocks, however, there is an opportunity to create cytology-derived FFPE TMAs [39] and accordingly perform high-throughput analyses on cytology specimens, which may sometimes represent the only sample available from patients.

      Clinicians and cytologists are sometimes faced with cytology samples that are non-diagnostic or inadequate for ancillary testing, which hinders appropriate patient management. This may result from various factors, including the nature of the lesion targeted for FNA, operator skill, and inadequate cellularity on the cell block. To improve the diagnostic yield, manufacturers of needles, especially for endoscopic ultrasound-guided procedures, have developed larger-gauge needles with new needle tip designs that extract “micro-cores.” The procedure and sample represent a hybrid – the motion to obtain the tissue fragments is the same as FNA biopsies, yet the needle size and design produce a “micro-core.” These samples typically contain mini tissue fragments in a core-shaped fragment of partially clotted blood. These FNA-derived micro-core biopsies represent a cross-roads between cytology and histology. There is some debate regarding the best method and laboratory ideally equipped to manage such specimens – either as cytology cell blocks or as histology biopsies. Since these samples, in addition to having visible tissue fragments, also contain cells dispersed in blood and liquid media, they are better managed by cytology procedures that will capture all of the cells by centrifugation