method – one that yields reproducible high cellularity, is not labor intensive and skill dependent, does not require the use of specific media, and is economical. Second, there is ongoing development and improvement of technologies in other aspects of medicine (e.g., minimally invasive procedures and molecular diagnostics) with demands to do more testing with less sample. As the traditional cell block methods have one or more shortcomings, there is ongoing interest in meeting the challenges and developing improved cell block methodologies to consistently provide high cellularity for diagnosis and ancillary testing. The basic techniques and attributes of traditional (e.g., plasma-thrombin, HistoGel) and newer methods, including the Cellient Automated Cell Block System, are discussed. The advantages of cell blocks, such as their ability to integrate with other existing technologies, such as whole-slide imaging, are also highlighted.
© 2020 S. Karger AG, Basel
Histology versus Cytology
The size of a tissue sample often determines whether the sample is processed via surgical pathology as a histology specimen, or via cytopathology as a cytology specimen. In general, if the tissue fragments are grossly visible, usually greater than 1 mm, they are processed as a histology sample. Dispersed cells, such as occurs in fluids, as well as samples with minute tissue fragments, such as in a fine-needle aspiration (FNA) biopsy, are typically processed as a cytology sample. The difference in size between histology and cytology specimens also dictates other steps in specimen management. Histology samples are often processed in the gross room, using tools such as forceps and scalpels, and slide preparation for immediate assessment (e.g., intraoperative consultation) is performed by generating a slide with a frozen section using a cryostat. Conversely, cytology samples are processed in a cytology laboratory, using tools and equipment such as a pipette, centrifuge, and vortex mixer, and immediate assessment is rendered from a slide smear or touch preparation.
While histology specimens are processed in a fairly standardized fashion across laboratories with formalin fixation, paraffin embedding, and hematoxylin and eosin (HE) staining, there is no consistent protocol for managing cytology specimens. For instance, there is no widely accepted standard for cytology specimen collection media, slide processing (e.g., smear, cytospin, liquid-based cytology; LBC), staining (e.g., Diff-Quik, Papanicolaou, and/or HE), and cell block preparation. Interestingly, the lack of standardization in cytology is partially a consequence of ongoing advancements in cytology (e.g., LBC), tissue acquisition techniques (e.g., minimally invasive procedures), and molecular diagnostics. New advances in cytology have not been matched with changes in standard operating procedures uniformly among laboratories. This dynamism requires continuous adaptation of cytology, including cell block processing.
Cell Blocks: Overview
Cell blocks are a point of convergence between cytology and histology. As a cytology specimen, a cell block is composed of single cells and minute tissue fragments, and similar to histology specimens, it is embedded in a paraffin block and can yield multiple slides for HE staining and ancillary studies. The value of a well-prepared cell block is to provide diagnostic information and adequate material for ancillary testing.
Cytology specimens from which cell blocks can be made include FNA and exfoliative samples; the latter includes gynecological (e.g., Pap tests) and non-gynecological specimens (e.g., effusions, bronchoalveolar lavage, cyst drainages, and urine). The vast majority of cell blocks are prepared from FNA and non-gynecological exfoliative specimens. In fact, FNAs are often routinely supplemented with cell blocks, especially in cases where malignancy is suspected clinically. Some institutions make cell blocks whenever a visible sediment is present, whilst others are more selective. For non-gynecological exfoliative samples, effusions represent the most common subtype to have accompanying cell blocks. Less frequently, they may be prepared from urine samples and Pap test specimens, especially to better characterize glandular lesions in Pap tests [1].
Cell blocks are prepared by concentrating the individual cells and small tissue fragments of a cytology specimen into a pellet. In this way, the sample is similar to a larger and more cohesive histology specimen. Once a pellet is formed, most cell block procedures will proceed by placing the sample in a tissue cassette and processing this sample as a histology specimen. Key steps for cell block procedures include rinsing aspirated material from an FNA into a medium, cytoconcentration, and pellet formation. These steps also represent points of divergence in procedures between laboratories.
Cell Blocks: Advantages
Cell blocks offer several advantages. By capturing small tissue fragments, the histologic architecture of a targeted lesion may be preserved. For instance, features such as intracellular bridging in squamous cell carcinoma or fibrovascular cores in papillary lesions are best appreciated on a cell block. Cell blocks also provide better examination of minute tissue fragments, which may be too thick to see on a smear or LBC preparation. A cell block can enhance the diagnostic yield of an FNA or cytology sample, especially when the cell block is made with residual or unused specimen remaining in a vial after making smears or LBC preparation slides [2–4]. A cell block can generate numerous recut slides for ancillary studies including cytochemical stains, immunohistochemical (IHC) stains and molecular testing by fluorescent in situ hybridization, polymerase chain reaction, and next-generation sequencing. Cell blocks are also a source of preparing additional recut slides for submission to another institution, enrollment into clinical trials, and future academic studies that do not compromise the original smears or LBC slides. Ordering ancillary studies from cell blocks, in contrast to using smears, integrates more easily into the existing workflow of the histology laboratory.
Although cell blocks offer many advantages, they are best used to complement rather than entirely replace other cytology preparations such as smears or LBC slides. A well-prepared smear or LBC sample can reveal cytologic detail that may not be well demonstrated in a cell block. For instance, Papanicolaou-stained cytology slides may highlight subtle nuclear features of a lepidic predominant lung adenocarcinoma, or cytoplasmic “orangeophilia” specific for squamous differentiation. Also, the lymphoglandular bodies seen in lymphoma cases or the tigroid background in seminoma or Ewing sarcoma best shown on Diff-Quik-stained cytology smears will not be appreciated in the cell block slides.
Cell Blocks: Current Methods
As a century old technique [5], cell blocks have long been an integral component of cytology samples; however, there has never been a widely accepted standard for how cell blocks are made. Indeed, there are several cell block-processing techniques, and more recently there has been great interest in maximizing their cellular yield. This is largely the result of a paradigm shift that has occurred under the moniker of “personalized medicine” with the advent of molecular diagnostics applied to minimally invasive biopsies to establish diagnoses and determine therapeutic options. This has led to an increased demand for cell block samples, which can be used for ancillary testing such as IHC staining and molecular sequencing assays. The prototypic example for this phenomenon is lung cancer, for which an FNA biopsy with a cell block is one of the common means to obtain tumor tissue for molecular characterization. Other tumors, from the pancreas or retroperitoneum, pose similar challenges, and reliance on cell blocks for ancillary testing has equally become significant.
Fig. 1. Common steps in cell block processing: most cell block processing methods involve centrifuging the specimen and decanting the supernatant, thereby leaving behind a cell pellet.