Cell blocks in cytopathology: a review of preparative methods,utility in diagnosis and role in ancillary studies

The cell block (CB) is a routine procedure in cytopathology that has gained importance because of its pivotal role in diagnosis and ancillary studies. There is no precise review in the published literature that deals with the various methods of preparation of CB, its utility in diagnosis, immunocytochemistry (ICC) or molecular testing, and its drawbacks. An extensive literature search on CB in cytology using internet search engines was performed for this review employing the following keywords: cell block, cytoblock, cytology, cytopathology, methods, preparation, fixatives, diagnostic yield, ancillary and molecular studies. Ever since its introduction more than a century ago, the CB technique has undergone numerous modifications to improve the quality of the procedure; however, the overall principle remains the same in each method. CBs can be prepared from virtually all varieties of cytological samples. In today's era of personalized medicine, cytological specimens, including CBs, augment the utility of cytological samples in analysing the molecular alterations as effectively as surgical biopsies or resection specimens. With the availability of molecular targeted therapy for many cancers, a large number of recent studies have used cytological material or CBs for molecular characterization. The various techniques of CB preparation with different fixatives, their advantages and limitations, and issues of diagnostic yield are discussed in this review.     

Angiosarcoma in FNA smears: diagnostic accuracy,morphology, immunocytochemistry and differential diagnoses

?. Pohar‐Marin?ek and J. Lamovec Angiosarcoma in FNA smears: diagnostic accuracy, morphology, immunocytochemistry and differential diagnoses Objective: The aim of our study was to analyse the diagnostic accuracy in recognizing angiosarcoma from fine needle aspiration (FNA) samples and to determine morphological features of angiosarcoma in cytology. Methods: FNA samples from 18 histologically confirmed angiosarcomas obtained between 1985 and 2009 were included in the study. Original cytological diagnoses were retrieved, smears reviewed and morphological features analysed: cellularity, smear pattern, cell morphology, contents of background. Outcome of immunocytochemistry was noted and additional reactions performed if material was available. Results: There were 13 primary angiosarcomas and five recurrent tumours; nine tumours were epithelioid. Twelve tumours were cytologically diagnosed as malignant, three as suspicious and three were judged unsatisfactory. Only two primary tumours were diagnosed as vascular. According to morphology, tumours were divided into those with predominantly epithelioid cells and those with predominantly spindle cells. Within these two groups were variations due to grade of tumour. Cytomorphology did not correlate well with histology in mixed and spindle cell types of angiosarcomas. Immunocytochemistry was applied in seven cases, specific vascular marker CD31 only twice at the time of diagnosis and three times retrospectively. Conclusions: Angiosarcomas are difficult to recognize on FNA smears when they lack the typical dual, spindle and epithelioid cell population and when they occur in internal organs where carcinomas are more common. Very few reliable data are available concerning specificity of CD31 on cytological material.     

Haemorrhagic cytology samples – how to get the best diagnostic results

OBJECTIVE: To describe and evaluate the value of a simple filtration technique for use in processing haemorrhagic samples for cytomorphological evaluation and immunocytochemistry. METHODS: One hundred and sixty haemorrhagic cytological samples (133 FNAs, 27 effusions) received in our laboratory from August 2002 to September 2005 were included in this study. After preparing two smears for diagnostic evaluation, the residual sample was suspended in 2 ml of a cell medium prepared in our laboratory. These primary haemorrhagic suspensions were filtered through disposable nylon filter devices and the cells deposited on the upper membrane surface were transferred into 2 ml of fresh cell medium. From all three fractions - primary cell suspension, filter deposit and filtrate - cytospins were prepared and stained by Giemsa or Papanicolaou methods. Cytospins were examined under the microscope for the presence of diagnostic cells, red blood cells (RBCs) and debris. Additional cytospins for immunocytochemistry were prepared at the cytopathologist's request. RESULTS: RBCs and debris were successfully removed in 142 out of 160 haemorrhagic samples (88%) by using this new filtration technique. In all these cases the tumour cells were well presented and allowed substantially improved cytomorphological evaluation. Immunocytochemical staining was performed on 112 filtered samples with three different markers per case on average. Filtration did not improve the quality of cytospins in 18/160 haemorrhagic samples, mostly attributable to insufficient numbers of diagnostic cells in the original samples. CONCLUSION: The presented filtration technique is very simple and quick. It substantially improves the quality of haemorrhagic samples for cytomorphological evaluation; moreover, the samples are well suited for multiple immunocytochemical stainings.     

Rapid KRAS, EGFR, BRAF and PIK3CA mutation analysis of fine needle aspirates from non-small-cell lung cancer using allele-specific qPCR

Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients.     

Update on: proteome analysis in thyroid pathology – part II: overview of technical and clinical enhancement of proteomic investigation of the thyroid lesions

ABSTRACT

Introduction: An accurate diagnostic classification of thyroid lesions remains an important clinical aspect that needs to be addressed in order to avoid ‘diagnostic’ thyroidectomies. Among the several ‘omics’ techniques, proteomics is playing a pivotal role in the search for diagnostic markers. In recent years, different approaches have been used, taking advantage of the technical improvements related to mass spectrometry that have occurred.

Areas covered: The review provides an update of the recent findings in diagnostic classification, in genetic definition and in the investigation of thyroid lesions based on different proteomics approaches and on different type of specimens: cytological, surgical and biofluid samples. A brief section will discuss how these findings can be integrated with those obtained by metabolomics investigations.

Expert commentary: Among the several proteomics approaches able to deepen our knowledge of the molecular alterations of the different thyroid lesions, MALDI-MSI is strongly emerging above all. In fact, MS-imaging has also been demonstrated to be capable of distinguishing thyroid lesions, based on their different molecular signatures, using cytological specimens. The possibility to use the material obtained by the fine needle aspiration makes MALDI-MSI a highly promising technology that could be implemented into the clinical and pathological units.     

Rapid Immunocytochemistry with Simple Heat-Induced Antigen Retrieval Technique for Improvement in the Quality of Cytological Diagnosis

Rapid immunocytochemistry (ICC) can improve the accuracy of intraoperative cytological diagnoses; however, it is usually applied without heat-induced antigen retrieval (HIAR). We established a HIAR method for rapid ICC and evaluated its efficacy and reliability. Rapidly fixed smear samples were immunostained using 35 antibodies. We compared the results of HIAR by boiling in a pot or heating in an electric kettle. The smears were incubated for 3 min with each primary antibody and immuno-enzyme polymer reagent, and for 1 min with diaminobenzidine solution. HIAR for 1 min using the kettle method yielded the best cellular integrity. For 32 out of the 35 antibodies, results achieved using rapid ICC within 11 min were comparable to that achieved using standard ICC. HIAR was essential for 13 antibodies. For two of the antibodies, HIAR was not required when standard ICC was applied, but consistent staining with rapid ICC was obtained only with HIAR. In conclusion, we established a rapid ICC procedure using a simple HIAR method, which allowed efficient immunostaining of a panel of antigens, including nuclear antigens, within only 11 min. The combined use of this rapid ICC technique with other staining techniques could be useful for improving intraoperative cytological diagnoses.     

Molecular diagnosis on tissues and cells: how it affects training and will affect practice in the future

C. Boyd and D. P. Boyle Molecular diagnosis on tissues and cells: how it affects training and will affect practice in the future On 25th November 2011, a symposium organized by the Royal College of Pathologists, entitled 'Molecular diagnosis on tissues and cells', took place in London. As trainees in histopathology and cytopathology, we were stimulated to consider the role that molecular biology is likely to play in future practice and how this is addressed by our own training. The symposium provided a basis for this article. Routine samples requiring molecular analysis are equally relevant to histopathologists and cytopathologists, and molecular biology laboratories are now using cytological as well as histological material for diagnostic testing, allowing different specimen types to be used as and when they are most appropriate. The most widely used types of molecular analysis in routine cellular pathology are EGFR testing in lung cancer, molecular testing of thyroid nodules, fluorescence in?situ hybridization testing of urine samples, clonality analysis in lymphoma testing, HER2 testing in breast and gastric cancer, KRAS testing in colorectal cancer, intraoperative assessment of breast cancer sentinel nodes, molecular testing of gastrointestinal stromal tumours and mismatch repair protein analysis. Of these, the majority in the UK are carried out on histopathology samples, although many are applicable to cytological samples if adequate material is obtained. We are particularly encouraged by the potential of molecular diagnostic cytology in traditionally difficult areas, such as intraoperative assessment. We believe that increasing reliance on molecular diagnostic techniques will also herald changes in training.     

Transmission of synovial sarcoma from a single multi-organ donor to three transplant recipients: case report

Quick and reliable testing of EGFR and KRAS is needed in non-small cell lung cancer (NSCLC) to ensure optimal decision-making for targeted therapy. The Idylla™ platform was designed for Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections but recently several studies were published that evaluated its potential for cytological specimens. This study aimed to validate the Idylla™ platform for the detection of EGFR/KRAS mutations in cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. The KRAS Idylla™ test were performed on 11 specimens with a known KRAS mutation. The EGFR Idylla™ test was performed on 18 specimens with a known primary EGFR mutation and 7 specimens with a primary EGFR-EGFR T790M resistance mutation combination. Concordant KRAS and primary EGFR mutations were detected for both KRAS and primary EGFR mutations. Samples with a total CQ value of < 26 could be considered negative. Samples with a total CQ value of > 26 could not be assessed (probability of false-negative). In specimens with a primary EGFR-EGFR T790M resistance mutation combination, 5/7 cases were not concordant. Our results confirm the conclusion of recent reports that the Idylla™EGFR assay is not suitable in a resistance to EGFR TKI setting, also not in our cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. KRAS and primary EGFR mutations were detected using the Idylla™ assays in virtually all cytological NSCLC samples. This analysis was rapid and time-saving compared to other mutation detection assays and may be useful if the amount of material is insufficient to perform a full set of molecular tests.     

Fine needle aspiration (FNA) of synovial sarcoma—a comparative histological–cytological study of 15 cases, including immunohistochemical, electron microscopic and cytogenetic examination and DNA‐ploidy analysis

A retrospective study of 25 FNAs (11 aspirates from primary tumours and 14 from recurrencies and metastases) from 15 synovial sarcomas was performed. The cytological findings were correlated with the histopathology and the value of immunohistochemical and electron microscopic examination as well as DNA‐ploidy and cytogenetic analysis for diagnosis were assessed. A reproducible cellular pattern with a reliable diagnosis of spindle cell sarcoma was possible provided that the aspirates were cell rich. However, a true biphasic pattern indicative of synovial sarcoma was only seen in one of the 25 specimens. Electron microscopic examination of the aspirates was a valuable adjunctive diagnostic method, whereas immunocytochemistry and DNA‐ploidy analysis were not. Immunohistochemical, electron microscopic and cytogenetic analysis were all valuable ancillary methods when performed on surgical specimens. Malignant haemangiopericytoma and fibrosarcoma were the most important differential diagnoses in the FNA specimens.     

Primary cell cultures arising from normal kidney and renal cell carcinoma retain the proteomic profile of corresponding tissues

Renal cell carcinoma (RCC) tissue is composed of a mixture of neoplastic and normal cells, which complicate proteome analysis. The aim of our study was to investigate whether it is feasible to establish primary cell cultures of RCC and of renal cortex maintaining the tissue phenotype along with a more homogeneous and enriched cytological material. Fourteen (82.3%) primary cultures from 17 surgical cases were established and characterized by morphology, growth rate, immunocytochemistry, and molecular analysis performed by Real-time PCR, Western blotting, two-dimensional electrophoresis (2-DE), and mass spectrometry. Cultures showed >90% cytokeratine-positive epithelial cells. In primary tumor cultures, the molecular phenotype of manganese superoxide dismutase and heat shock protein 27 was the same as that found in tumor tissues with overexpression and increased number of isoforms. Moreover, 27 out 28 specific proteins and their isoforms, present in spots excised from 2-DE gel of cortex or RCC cultures, corresponded to those identified on the 2-DE tissue cortex reference map, suggesting that these primary cultures retain the proteomic profile of the corresponding tissues.     

Hodgkin's disease: diagnostic accuracy of fine needle aspiration; a report based on 62 consecutive cases

We report on our series of 62 cases occurring between January 1977 and December 1990, which were diagnosed as Hodgkin's disease by fine needle aspiration (FNA) samples. the overall accuracy of the cytological diagnosis was high, with only four incorrect diagnoses and a positive predictive value of 93.5%. the value of FNA as a first level diagnostic technique in the screening of lymphadenopathies is discussed, as well as the limitations and pitfalls of the cytological diagnosis.     

Diagnostic value of immunohistochemistry in lesions of the endocrine system

The aim of immunohistochemistry and immunocytochemistry is to reveal specific antigens in cells and tissue samples. Those techniques are based on an antigen-antibody reaction and visualization of its product in microscopic examination. The precursor of this new diagnostic procedure was an immunofluorescent reaction in frozen tissue samples performed by Albert Coons in 1940. Then the immunohistochemical techniques were perfected to increase sensitivity and specificity. Currently it is hard to imagine a modern pathological examination without immunohistochemistry. At the end of XXth century it was believed that 75% of cases is possible to be diagnosed due to immunohistochemical stains. Microscopic examination of endocrine glands tissue samples is extremely difficult because of coexistence of the presence of neoplasms and endocrine dysfunction. It is necessary to establish the type of hormones in the cells of the endocrine system lesions to make a proper diagnosis. Thanks to the use of antibodies against hormones and its precursors it becomes possible. At present most of the antigens are easily detected in both: formalin fixed paraffin embedded tissues samples and ethanol fixed cytological smears so immunohistochemocal and immunocytological stains can be a part of routine diagnostic procedures in pathology. However most of the biologically active substances are revealed in many organs and tissues and it is necessary to perform a satisfactory immunohistochemical panel to be sure the diagnosis. It is important to notice that there is no need to make a wide panel of antibodies in all of the cases and the economical aspect of examination is also important. Of course immunohistochemistry sometimes is the guarantee of proper diagnosis but in some cases too wide panel of antibodies can be a loss for the patient and for medical department. We discussed the proper diagnostic procedures and immunohistochemical profile in pathological lesions of endocrine system (thyroid and adrenal gland, adenohypophysis, neuroendocrine tumours and some hormones-secreting tumours of gonads).     

Is CSF cytology a useful diagnostic procedure in staging paediatric CNS tumours?

Objectives:  To evaluate whether there are any factors that predict malignant cells being found in paediatric cerebrospinal fluid (CSF) samples. To determine whether CSF provides useful staging information not provided by magnetic resonance imaging (MRI) in paediatric patients with primary central nervous system (CNS) malignancy.
Methods:  We compared the CSF cytology and spinal MRI staging results in paediatric patients with primary CNS malignancy at a UK tertiary referral centre, over a decade.
Results:  Of 159 CSF samples, 72 samples were from 72 patients with primary CNS malignancy with spinal MRI available for comparison. Eight of these 72 had positive cytology (seven malignant and one suspicious). All had a high clinical suspicion of tumour at the time of sampling. Of the 72 patients, only two had evidence of CSF spread on MRI spinal staging and CSF cytology; ten had MRI without cytological evidence and six had cytological without MRI evidence.
Conclusions:  In paediatric patients with primary CNS tumours, CSF cytology provides useful staging information. Spinal MRI alone may miss some patients with CSF spread who would be identified with CSF cytology.     

Extraction of DNA from exfoliative cytology specimens and its suitability for analysis by the polymerase chain reaction

The extraction of DNA from archival exfoliative cytology samples would allow the molecular biological analysis of this readily available material using the polymerase chain reaction (PCR). We have quantitatively and qualitatively studied the extraction of DNA from a variety of cytological preparations. For both fresh and archival cervical smears, overnight incubation with proteinase K produces high yields of high molecular weight DNA, but simply boiling the samples produces DNA suitable for PCR amplification of a single copy gene. Increasing the proteinase K incubation to several days allows the extraction of DNA from fixed and stained archival cytology slides from a variety of sites. The extracted DNA was again suitable for PCR analysis. Fresh and archival cytological material can be utilized for molecular biological study of disease processes using PCR. Archival cytological material is probably the best source of DNA and RNA after stored frozen tissue.     

The evaluation of liquid-based ''Cyto-SEDTM'' cytology of bronchioalveolar lavage specimens in the diagnosis of pulmonary neoplasia against conventional direct smears

Historically, bronchioalveolar lavage (BAL) samples have been prepared by a direct smear (DS) technique. Recent advances in liquid-based cytology have led to a revolution in cytological specimen preparation. Cyto-SED system (CS) is a manual liquid-based cytology system, designed for small-scale use. A total of 137 samples from patients with radiographically detectable lesions underwent BAL procedures at Papworth Hospital NHS Trust over a 4-month period. After preparation for diagnostic purposes with the DS method, the remaining sample was prepared using the CS system. The slides produced were allocated a blind study number and screened by three independent screeners. The cellular morphology was well preserved and comparable between both techniques. Of the 137 patients, 38% were confirmed as malignant by cytology or histology; 71% of these malignant diagnosis were confirmed by the DS technique and 91% confirmed by the CS. The results demonstrate that the CS is a viable alternative to the DS technique. The cytological detail is clearly defined without a loss of three-dimensional information, thus aiding the differential diagnosis of malignancy. Cyto-SED cytology system yields a higher diagnostic accuracy than the conventional direct smear technique without compromising on cytological detail.     

COS cells expression cloning of tyrosine-phosphorylated proteins by immunocytochemistry.

Tyrosine phosphorylation is an important post-translational modification of proteins, essential in many aspects of the cell economy, particularly in signal transduction pathways. Despite the importance of protein tyrosine phosphorylation, the approaches available for molecular cloning remain limited. We have developed a COS cell-based eukaryotic expression cloning procedure for phosphotyrosine-containing proteins by immunocytochemistry of cell monolayers. The approach takes advantage of the low basal levels of tyrosine phosphorylated, robust transient expression, availability of specific antibodies against tyrosine-phosphorylated residues, and rescue of episomal DNA after immunocytochemistry. The technique is validated by cloning the rat proto-oncogene c-fgr in its tyrosine-phosphorylated form out of a rat kidney cDNA library containing over 10(6) primary recombinants. This technique set the grounds for expression cloning of tyrosine-phosphorylated proteins in eukaryotic cells, and it is anticipated that further modifications and refinements will allow the identification of protein tyrosine phosphatase substrates.     

Technical aspects of the use of cytopathological specimens for diagnosis and predictive testing in malignant epithelial neoplasms of the lung

Lung cancer is a leading cause of cancer mortality worldwide but recent years have seen a rapidly rising proportion of cases of advanced non-small cell carcinoma amenable to increasingly targeted therapy, initially based on the differential response to systemic treatment of tumours of squamous or glandular differentiation. In two-thirds of the cases, where patients present with advanced disease, both primary pathological diagnosis and biomarker testing is based on small biopsies and cytopathological specimens. The framework of this article is an overview of the technical aspect of each stage of the specimen pathway with emphasis on maximising potential for success when using small cytology samples. It brings together the current literature addressing pre-analytical and analytical aspects of specimen acquisition, performing rapid onsite evaluation, and undertaking diagnostic and predictive testing using immunocytochemistry and molecular platforms. The advantages and drawbacks of performing analysis on cell block and non-cell block specimen preparations is discussed.