Role of immunocytochemistry in cervical cancer screening.

The cervical cancer screening has been based conventionally on cytologic analysis. With advances in understanding the role of human papillomavirus, cotesting has been applied. But most of the patients subjected to colposcopy did not benefit, except in cases with HSIL [high-grade squamous intraepithelial lesion] cytology. Because of this, a step to increase the sensitivity to detect cancers and pre-cancers but with higher specificity with minimal overdiagnosis leading to prevention of unindicated cervical biopsies is highly desired. Such triaging step in cases with abnormal screening results is expected to minimize invasive interventions because of low false positivity. With availability of methodologies leading to quantitatively and qualitatively enhanced cell-blocks from residual liquid based cytology specimens, immunostaining can be performed for multiple immunomarkers with potential objectivity to triage initial screening test results. This is enhanced further with inclusion of AV marker in the cell-blocks and application of SCIP (subtractive coordinate immunoreactivity pattern) approach. The cell-blocks are also resource for performing other ancillary studies including molecular pathology and proteomics/metabolomics as potential tests in future. This review explores application of residual liquid based cytology specimen for cell-blocking with application of ancillary studies in algorithmic manner as adjunct to ASCCP management guidelines for improved patient care.

Atypical glandular cells (AGC): Cytology of glandular lesions of the uterine cervix.

The Pap smear is a well-known screening tool for squamous lesions of the uterine cervix. However, its screening role in glandular lesions is less effective. The incidence of squamous cell carcinoma of the cervix has dramatically decreased with the advent of Pap smear and recent understanding related to HPV carcinogenesis of cervical cancers including the advent of HPV vaccines. However, in recent years, the incidence of glandular abnormalities, diagnosed on Pap smears, has increased with greater sensitivity and precision. The incidence of atypical glandular cells (AGC) is approximately 0.18-0.74% of all cervical smears with a reported prevalence of 2.5% among all Pap smears. A high degree of suspicion, good clinical history, and the presence of diagnostic cytomorphological findings are essential for the proper interpretation of glandular cell abnormalities. A methodical approach to evaluate Pap smear greatly helps interpretation and avoids the diagnostic pitfalls. The Bethesda System for reporting cervical cytology has categorized glandular cell abnormalities into various categories as follows: Endocervical adenocarcinoma in situ (AIS)Atypical glandular cells (AGCs) Endocervical cells: a1 NOS or specify in comments; a2 Favor neoplasticEndometrial cells: NOS or specify in comments Adenocarcinoma (AdCa) EndocervicalEndometrialExtrauterineNOS Subtle differences in quantitative and qualitative cytologic features are essential for distinguishing one category from another. In this chapter, we highlight an organized approach for the interpretation of glandular abnormalities in Pap smear for our readers. This is an overview of the Bethesda categories, the reason for classification, and differential diagnosis with key characteristic features. An approach to the methodical evaluation of hyperchromatic crowded groups is discussed with key cytomorphologic differences. An algorithmic approach is suggested to facilitate the interpretation of various AGC categories.

Serous fluid: Reactive conditions.

This chapter highlights the steps that would help to analyze any fluid. It highlights importance of knowing gross analysis of fluid along with biochemical information. These parameters along with clinical information are very important in arriving at a differential diagnosis. Morphologic appearances in the fluid can often become challenging and occasionally reactive conditions can reveal changes that may mimic malignancies. This chapter provides not only a framework of approach to assessment of fluid cytology but also shows how to distinguish some of the challenging reactive conditions from the diagnosis of carcinoma. The chapter also utilizes two cases to demonstrate approach to reactive conditions. This review article will be incorporated finally as one of the chapters in CMAS (CytoJournal Monograph/Atlas Series) #2. It is modified slightly from the chapter by the initial authors in the first edition of Cytopathologic Diagnosis of Serous Fluids.

Immunocytochemistry of effusions: Processing and commonly used immunomarkers.

Definitive cytopathological interpretation of some of the effusion fluids may not be possible based on cytomorphological evaluation alone. As discussed in other reviews, this is due to various reasons specifically applicable to effusion fluids including remarkably wide morphologic spectrum of reactive mesothelial cells overlapping with some well to moderately differentiated metastatic carcinoma. The challenge is subject to various factors including level of interpreter training or experience, institutional demographics (such as type of prevalent diseases, predominant sex and age group), technical advances in ancillary support, and expertise in cytopreparatory processing. In such cases immunohistochemistry performed on cell-block sections is simple objective adjunct with or without other ancillary techniques. Ongoing increase in number of immunomarkers along with rabbit monoclonal antibodies with relatively higher affinity is further refining this field. SCIP (subtractive coordinate immunoreactivity pattern) approach, discussed as separate dedicated review article, facilitates refined interpretation of immunoreactivity pattern in coordinate manner on various serial sections of cell-blocks. However, many variables such as delay after specimen collection, specimen processing related factors including fixation and storage; ambient conditions under which paraffin blocks are archived (for retrospective testing); antigen retrieval method; duration of antigen retrieval step; antibody clone and dilution; and antibody application time are common with application of immunohistochemistry in other areas. This review is dedicated to highlight technical aspects including processing of effusion specimens for optimum immunocytochemical evaluation along with commonly used immunomarkers in effusion cytopathology. This review focuses on the technical and general information about various immunomarkers.

Collection and processing of effusion fluids for cytopathologic evaluation.

Accumulation of fluid in serous cavities as effusions may have to be drained for therapeutic and diagnostic indications. As compared to many other procedures, the technicality of paracentesis procedures is relatively easy. As a result, effusion fluids comprise a significant proportion of specimens in most of the laboratories, including those in community settings. Because of relative complexities in the cytopathologic evaluation of effusion fluids, application of appropriately standardized protocol is critical for achieving optimum results by applying standardized steps from handling of specimens during the initial stages of collection to the final interpretation phase. Understanding various limitations and challenges during collection and processing phases by all the personnel involved, including clinicians, pathologists, and technologists is critical for optimum diagnostic yield. This review highlights various cytopreparatory techniques applicable to effusion cytology in one place. It is organized by projecting the details predominantly in the form of different tables and figures including summary of the recommended protocols with reagents and stains used. Also included is a sample of cytopathology report based on the approach discussed in this series.

Immunocytochemistry of effusion fluids: Introduction to SCIP approach.

Due to the remarkably wide morphologic spectrum of reactive mesothelial cells, some of the effusion fluids may be difficult to interpret with objective certainty by cytomorphology alone. Cytomorphology of well to moderately differentiated adenocarcinomas (responsible for the bulk of malignant effusions) may overlap with floridly reactive mesothelial cells. Even mesotheliomas including diffuse malignant epithelioid mesothelioma, are usually cytomorphologically bland without unequivocal features of malignancy. The intensity of challenge depends on the interpreter's training or experience level, institutional demographics of patients (such as type of prevalent diseases, predominant sex and age group), technical support, and quality of cytopreparatory processing. In general immunocytochemistry is valuable adjunct to facilitate objective interpretation with or without other ancillary techniques as indicated. An increasing number of immunomarkers is further refining the contribution of immunohistochemistry to this field. However, application of immunohistochemistry to effusion fluids is relatively challenging because of many variables. Multiple factors such as delay after specimen collection, specimen processing related factors including fixation and storage; ambient conditions under which paraffin blocks are archived (for retrospective testing); antigen retrieval method; duration of antigen retrieval step; antibody clone and dilution; and antibody application time are identical to application of immunohistochemistry in other areas. The significant challenge related to the potential compromization of the immunoreactivity pattern due to exposure to non-formalin fixatives / reagents is also applicable to effusion fluid specimens. The immunoreactivity results would be compared and corelated with cumulative metadata based on the reported studies performed and validated on formalin-fixed paraffin-embedded tissue sections. Deviating from such protocols may lead to suboptimal results, which is not uncommon in clinical practice with potential compromization of patient care and related liability. Because of this, it is critical to perform immunocytochemistry on formalin-fixed cell-block sections only. In addition, unless the interpretation criteria for immunohistochemical evaluation of effusion fluids are not modified specifically, it may not be productive in resolving some challenging cases. However, this aspect is not well elaborated in the literature. A basic and critical challenge is finding and locating the cells of interest in cell-block sections of effusion fluids. A unique approach is to choose a fundamental immunopanel which highlight the mesothelial and inflammatory cells in reactive effusion fluids to create the basic map. This allows detection of a 'second-foreign' population which can be immunocharacterized further with the help of subtractive coordinate immunoreactivity pattern (SCIP) approach elaborated here.

Metastatic Carcinoma in Effusions.

Serous cavity may be involved by any neoplasm, including very rare examples of involvement by central nervous system tumors leading to a malignant effusion. The serous cavity lining is rich in lymphatics with lymphatic lacunae opening directly through narrow gaps (stoma) in the lining. Carcinomas mainly metastasize to serosa via the lymphatic vessels, which may be blocked leading to effusion. Primary carcinomas of organs such as lung, intestines, liver, ovary, etc., lined by serosal membranes may spread by direct extension, resulting in malignant effusions. As standard of practice, unless specified, cytopathologic examination of serous effusions implies detection of malignant cells. As compared to a surgical biopsy from a small focal area of an extensive serosal surface, effusion fluid from respective cavity exfoliates the cells from the entire serosal surface with minimal chance of sampling artifact. Because of this, effusion fluid cytology generally provides a higher diagnostic yield as compared to biopsy of the serous lining, as demonstrated by some studies. However, various challenges related to effusion fluid cytology makes the interpretation of effusion fluid cytology a field with potential misinterpretations, especially for those without proper experience or training. Developing and following a methodical approach is important for appropriate cytologic examination of effusion fluids. Proper approach may achieve definitive interpretation even without ancillary tests. However, lack of appropriate approach and processing may introduce a significant variation in interpretation due to combination of well-recognized diagnostic pitfalls, which may lead to lower reproducibility and even serious misinterpretations. Current review discusses in brief appropriate approach to processing and evaluating effusion fluid cytology for metastatic carcinoma. At general level, this is comparable to that of other specimens; however, it is critical to modify with reference to the limitations associated with effusion cytology.

Impact of cytopathology authors work: Comparative analysis based on Open-access cytopathology publications versus non-Open-access conventional publications.

OBJECTIVES: Open access (OA) is based on a set of principles and a range of practices through which fruits of research are distributed online, free of cost, or other access barriers. According to the 2001 definition, OA publications are without barriers to copy or reuse with free access to readers. Some studies have reported higher rates of citation for OA publications. In this study, we analyzed the citation rates of OA and traditional nonOA (with or without free access) publications for authors publishing in the subspecialty of cytopathology during 2010-2015. MATERIAL AND METHODS: We observed and compared citation patterns for authors who had published in both OA and traditional non-OA, peer-reviewed, scientific, cytopathology journals. Thirty authors were randomly selected with criteria of publishing a total of at least five cytopathology articles over 2010-2015. Number of citations per article (CPA) (during 2010-2015) for OA publications (in CytoJournal and Journal of Cytology) and non-OA publications (in Diagnostic Cytopathology, Cytopathology, Acta Cytologica, Journal of American of Cytopathology, and Indian Journal of Pathology and Microbiology) was collected and compared statistically using two-tailed Student's t-est. The data were collected manually through science citation analysis sites, mostly Google Scholar. RESULTS: Thirty authors published 579 cytopathology articles in OA and non-OA journals. Average CPA for OA publications was 26.64. This was 11.35 higher than the average CPA) of non-OA conventional with subscription cytopathology journals (74% increase) and 11.76 higher than the average CPA of conventional cytopathology non-OA journal articles with free access (79% increase). These differences were statistically significantly with P < 0.05. CONCLUSION: We observed that the cytopathology publications in the OA journal attained a higher rate of CPA than the publications in the traditional non-OA journals in the field of cytopathology during 2010-2015.

Squamous intraepithelial lesions (SIL: LSIL, HSIL, ASCUS, ASC-H, LSIL-H) of Uterine Cervix and Bethesda System.

For every 100,000 women in the United States, eight new cervical cancer cases and two deaths are reported as per the most recent (2017) Center of Disease Control and Prevention statistics. Of all the gynecologic cancers (ovary, uterus, cervix, vagina, and vulva), only cervical cancer has a screening test. Cervical Pap test (or Pap smear) is the best screening method for cervical precancerous lesions and is best reported using a unified and a well-established reporting system like The Bethesda System. In this system, "Epithelial cell abnormality: Squamous" includes squamous intraepithelial lesion (SIL) category which encompasses a spectrum of squamous cell lesions starting from the precancerous lesions of low-grade SIL (LSIL) to high-grade SIL (HSIL), and ultimately invasive squamous cell carcinoma. However, depending on the qualitative and quantitative limitations with the specimen, some equivocal morphological features suggestive of squamous cell abnormality may fall under equivocal category: "Atypical Squamous Cells" (ASCs), which are subdivided into two categories; "Atypical Squamous Cells of Undetermined Significance" (ASC-US) or "Atypical Squamous Cells, HSIL cannot be excluded" (ASC-H), based on the suspected underlying lesion LSIL versus HSIL, respectively. This review provides the key cytologic features that distinguish Bethesda squamous categories from other important entities, using algorithmic approach and illustrations of common cytomorphologic patterns for clear identification of those entities in practice. The important mimickers which may be considered during the differential interpretation of SIL are discussed and presented here in a brief cytomorphologic review.

Pleural effusion in hematological pathology.

A 51-year-old male with a history of chronic myelomonocytic leukemia-2 (CMML-2) presented with fatigue, night sweats, dyspnea, and right-sided chest pain exacerbated by deep breath. Computed tomography scan demonstrated right-sided pleural effusion with atelectasis. Pleural fluid cytology showed reactive mesothelial cells mixed with atypical cells [Figure 1]. The immunostains are performed using the SCIP approach.[1] The atypical cells were immunoreactive for vimentin, CD68, and CD163, while non-immunoreactive for cytokeratin, calretinin, BerEP4, and MOC31.

Cell-blocks and other ancillary studies (including molecular genetic tests and proteomics).

Many types of elective ancillary tests may be required to support the cytopathologic interpretations. Most of these tests can be performed on cell-blocks of different cytology specimens. The cell-block sections can be used for almost any special stains including various histochemistry stains and for special stains for different microorganisms including fungi, Pneumocystis jirovecii (carinii), and various organisms including acid-fast organisms similar to the surgical biopsy specimens. Similarly, in addition to immunochemistry, different molecular tests can be performed on cell-blocks. Molecular tests broadly can be divided into two main types Molecular genetic tests and Proteomics.

Cell-blocks and immunohistochemistry.

The interpretation of results on immunostained cell-block sections has to be compared with the cumulative published data derived predominantly from formalin-fixed paraffin-embedded (FFPE) tissue sections. Because of this, it is important to recognize that the fixation and processing protocol should not be different from the routinely processed FFPE surgical pathology tissue. Exposure to non-formalin fixatives or reagents may interfere with the diagnostic immunoreactivity pattern. The immunoprofile observed on such cell-blocks, which are not processed in a manner similar to the surgical pathology specimens, may not be representative resulting in aberrant results. The field of immunohistochemistry (IHC) is advancing continuously with the standardization of many immunomarkers. A variety of technical advances such as multiplex IHC with refined methodologies and automation is increasing its role in clinical applications. The recent addition of rabbit monoclonal antibodies has further improved sensitivity. As compared to the mouse monoclonal antibodies, the rabbit monoclonal antibodies have 10 to 100 fold higher antigen affinity. Most of the scenarios involve the evaluation of coordinate immunostaining patterns in cell-blocks with relatively scant diagnostic material without proper orientation which is usually retained in most of the surgical pathology specimens. These challenges are addressed if cell-blocks are prepared with some dedicated methodologies such as NextGen CelBloking™ (NGCB) kits. Cell-blocks prepared by NGCB kits also facilitate the easy application of the SCIP (subtractive coordinate immunoreactivity pattern) approach for proper evaluation of coordinate immunoreactivity. Various cell-block and IHC-related issues are discussed in detail.