Perfecting the fine-needle aspirate cell block

Authors

  • Neda Kalhor,

  • Ignacio I. Wistuba


  • Cytopathology Help Desk represents the opinions and views of the author and does not reflect any policy or opinion of the American Cancer Society, Cancer Cytopathology, or Wiley unless this is clearly specified.

History of the Cell Block in Cytopathology

The cell block (CB) technique has been in use for more than a century. The first report of this technique was made by Bahrenburg in 1896.1 He described a novel method that was applied to large quantity ascitic fluid in 2 patients, which enabled him to process the fluid into histologic sections. Dr. Bahrenburg's technique consisted of allowing the fluid to stand and clot spontaneously for 24 hours, followed by pouring off the supernatant fluid and allowing the sediment to be hardened by addition of alcohol to obtain a firm and hard tissue mass that was subsequently embedded in celloidin. The tissue block was then cut and stained similar to histologic sections. Following this report, various CB techniques have been developed over the years that vary in scope and the type of fixatives, processing, and embedding techniques used. Some of the most common techniques include inverted filter sedimentation, collodion bag, thrombin method, simple sedimentation, and Millipore filtration, among others.

Accuracy and Value of CB Material

The value of CB as a complementary diagnostic material for morphologic evaluation is well recognized. The architectural details of the tissue may be better preserved in the CB, and the hematoxylin and eosin stain is familiar to all pathologists. Few studies have compared the diagnostic value of CBs with other cytologic preparations such as direct smears and ThinPreps.2–6 These studies show that the sensitivity of the CBs, although slightly lower, are comparable to smears and ThinPreps. More importantly, the overall sensitivity improves when these techniques are applied together.

Creating Formalin-Fixed Tissue From Cytologic Fine-Needle Aspirates

Although the role of CB in providing additional material to resolve diagnostic difficulty is valuable, the main effectiveness of the CB technique lies in providing formalin-fixed paraffin-embedded (FFPE) tissue suitable for a variety of ancillary studies such as immunohistochemical studies and molecular testing. Some of the recent work on cytologic smears and CBs have sought to highlight the fact that these materials can be used for molecular analysis as valuable alternatives for surgical specimens.7,8 In fact, cytologic specimens offer several advantages over surgical specimens: 1) onsite immediate assessment is primarily possible for cytologic preparations, and that has been shown to improve diagnostic yield and specimen adequacy; 2) the ability to redirect needle during fine-needle aspiration (FNA) procedure with immediate assessment is a well-known advantage over needle core biopsy; 3) the cytologic samples are frequently purer for molecular analysis due to lower contamination of non-neoplastic cells, such as stromal cells; 4) direct smears display the entire nucleus of cells in contrast to a fraction of nuclear content in histologic sections from FFPE tissue blocks; and 5) superior quality genomic nucleic acid is present in direct smears due to lack of exposure to formalin fixation or paraffin embedding.9 There are also some disadvantages for the use of cytology material for molecular studies, including that the direct smears are not recoverable once they are used for testing, not all specimens are suitable for CB preparation, and a significant volume of cell block preparations lack sufficient tumor cellularity for testing.

Molecular Testing in FNA Cytopathology

Molecular profiling of tumor has become crucial for selection of treatment and monitoring molecular evolution of the tumor during the course of therapy. Currently, the majority of biomarkers required for targeted therapy in different types of cancer such as lung, colorectal, thyroid, and melanoma are oncogene mutations, gene fusions, gene copy number gains, and protein overexpression that can be assessed by methodologies such as polymerase chain reaction (PCR)-based sequencing, fluorescence in situ hybridization, quantitative PCR, and immunohistochemistry. All these techniques can be successfully applied to archived smears as well as CBs; however, FFPE tissue is the most commonly required form of tissue for testing in the vast majority of the pathology laboratories. In many circumstances, effusion specimen, FNA, or endoscopic bronchial ultrasound-guided biopsies may be the only specimen available for molecular testing, and the application of molecular markers to these materials will help prevent unnecessary, repeat biopsy procedures. The clinical utility of cytology specimens as a reliable source for molecular testing in patients with lung cancer has been validated recently.7,8 PCR can be performed using cells scraped from cellular smears, or from 3- to 10-mm tissue sections of cell blocks prepared from effusion or FNA samples. Billah et al7 reported successful EGFR and KRAS mutational analyses performed on various cytology specimens, with an overall specimen insufficiency rate of only 6.2%. The vast majority of the specimens included in this study were CB preparations (55%).

The Preferred Method of the Authors

At The University of Texas MD Anderson Cancer Center, Houston, Texas, CBs are routinely made on body cavity fluid or FNAs with sufficient tissue particles that can be prepared to CB. Following smear preparations, any excessive material is submitted in the rinse (RPMI 1640, Gibco brand; Invitrogen by Life Technologies, Carlsbad, California) for CB preparation. If ancillary studies are indicated, separate passes are recommended to ensure sufficient tissue for testing. The rinse is then centrifuged at 1500 rpm for 10 minutes. After decanting the supernatant, the remaining centrifugate is mixed with an equal amount of 10% formalin and 95% ethanol and again centrifuged at 1500 rpm for 10 minutes. The button of concentrated material is folded in sharkskin filter paper and placed in a tissue cassette and placed in 10% formalin and processed in the histology laboratory. For scanty specimens, we use the collodion CB method to enhance tissue collection. A valid comparison of the efficacy of various methods of CB preparation is very difficult due to lack of uniform methodology and vast differences in technical details. There is also a wide selection of fixatives available for CB preparation. However, some of these fixatives have been disapproved due to health hazards (ie, B5 solution, which contains mercury) or interference with DNA preservation for molecular testing (B5 and Bouin's solution). Ethanol is optimal for preservation of the nucleic acid; therefore, CB preparations using alcohol-based fixatives may have superior nucleic acid quality compared with FFPE surgical specimens.

Proper tissue triaging and handling has become an important aspect of the pathologist's daily practice. To optimize this process and to avoid subjecting the patients to additional unnecessary sampling, a pathologist needs to develop knowledge and understanding of pros and cons for different techniques, appropriate type of tissue needed for a specific test, and amount of tumor needed for that particular methodology. The methodologies and required tissue preparation available may vary in different practices and laboratories, and it is important for the pathologist to be familiar with them. The most commonly used technique for gene mutation analysis is PCR-based sequencing that can be applied to both cytologic and surgical preparations. The recommended amount of tumor cells in a sample qualified for testing by these techniques is a minimum of 20% tumor content and at least 500 tumor cells. However, with the development of new technologies for large-scale sequencing, such as next-generation sequencing of DNA and RNA, which has a significantly higher sensitivity, the same amount of tumor can be used for analysis of a 400-gene panel.

We recommend using smaller (22- to 25-gauge) needles because a larger needle causes more tissue injury and bleeding and does not yield more favorable tumor cellularity.10–12

Adequate sampling generally requires several passes, frequently a minimum of 3. An on-site cytopathologist performing immediate evaluation for specimen adequacy has been shown to improve overall diagnostic yield and facilitate effective specimen triage. Archived cytology smears up to 16 years old have been shown to yield sufficient quantities of DNA suitable for molecular testing.13 However, new tissue samplings are often recommended due to molecular alterations within tumor genomics associated with tumor progression following treatment, including molecular mechanisms of resistance to a given therapy.

Conclusions

In summary, there is potential for use of cytology specimens for molecular analysis in this era of biomarker-based targeted therapy. The role of cytology remains to be adequately recognized by pathologists, clinicians, hospitals, and researchers. More investigation is needed to determine ways of improving specimen processing to streamline the use of cytology material and to validate outlines on quality control.

Biographical Information

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Neda Kalhor MD

Neda Kalhor, MD, is an Assistant Professor in the sections of Thoracic Pathology and Cytopathology at The University of Texas MD Anderson Cancer Center, Houston, Texas. She is board certified in Anatomic and Clinical Pathology and Cytopathology. She is the leading clinical pathologist in the MD Anderson lung cancer BATTLE trials and a coinvestigator in several funded grants and research agreements.

Biographical Information

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Ignacio I. Wistuba MD

Ignacio I. Wistuba, MD, is the Jay and Lori Eisenberg Professor and Chair of the Department of Translational Molecular Pathology, with a joint appointment in the Department of Thoracic/Head and Neck Medical Oncology, at The University of Texas MD Anderson Cancer Center. He currently serves as the pathologist of the Lung Cancer Committee for the Southwestern Oncology Group, the Lung Cancer Mutation Consortium, and member of the Pathology Panel of the International Association for the Study of Lung Cancer. He also serves as Senior Editor of Cancer Prevention Research and on the Editorial Boards of Clinical Cancer Research and Journal of Clinical Oncology.

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