Shared Perspectives from Cytology and Microbiology

Cytology, also known as cytopathology, and microbiology laboratories reside in hospitals, clinics, or free-standing laboratories, in which scientists are employed to diagnose many medical conditions, including infection, inflammation, or cancer. There is overlap in the types of clinical samples shared by cytology and microbiology laboratories. Specimens include swabs, sputum, and fine-needle biopsy specimens or aspirates. When laboratories share clinical samples, there can be similarities and differences in the proper transport media and preservatives used. When specimens or functions are shared, there is an opportunity for each laboratory to learn from each other. Increased awareness of the roles and processes in cytology and microbiology can decrease the number of lost or improperly preserved specimens. Improved communication and collaboration between laboratory sections can increase diagnostic accuracy and reduce patient harm. This review summarizes laboratory career choices, sample collection devices, shared specimens, and preservatives associated with shared specimens. Cytology and microbiology laboratories can work together to improve pre-analytic processes and diagnostic accuracy.     

Molecular techniques in cytopathology practice

In the last decade, new molecular techniques were introduced into pathology laboratories. Cytology also benefited from the innovations emerging from this new era. Molecular cytopathology (MCP) can be defined as molecular studies applied on all types of cytological specimens, namely gynaecology cytology, exfoliative non- gynaecology cytology and fine needle aspirates. The development of many new ancillary techniques has paralleled the emergence of clinical cytology as a major diagnostic specialty. Clinical applications of these techniques have been growing in the last decade. The widespread acceptance of liquid-based systems in gynaecological cytology emphasises the relation between cells and molecules. The increased use of morphology and molecular biology in human papillomavirus-induced lesions for example, showed the potential to optimise, in one single brushed sample, diagnosis and research. Cytology samples from serous effusions, the pulmonary tree, urine, and aspirations, among others, are now likely to be studied by different molecular techniques for diagnosis, prognosis, or even assessment of therapeutic targets. In this review, the main published results concerning the application of molecular techniques in different fields of cytopathology are highlighted, and their applications discussed.     

Discrepancy analysis, communication, and feedback for cytotechnologist quality improvement of nongynecologic cytopathology

The Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) detail the requirements for the cytotechnologist (CT) who evaluates gynecologic cytopathology specimens. However, the role of the CT in nongynecologic cytopathology is not clearly defined. Furthermore, non gynecologic cytopathology cases are diverse and the screening, interpretative, and diagnostic issues may be quite different from the gynecologic cases. At our institution, the CT and pathologist review nongynecologic cytopathology cases. Since CLIA '88 does not require the CT to screen nongynecologic cytopathology cases, there are few guidelines for quality assessment or quality improvement for the CT regarding nongynecologic cytopathology cases. To provide better understanding of the expectations of the CT and the needs of the pathologist, we developed a system comparing the CT's interpretation to the pathologist's interpretation as a means for enhanced communication and feedback. Using our Laboratory Information System (LIS), we generate a daily report that lists all cases with discrepancy in diagnoses between the CT and pathologist. The general supervisor reviews this report for diagnostic discrepancy in each case. To determine the degree of discrepancy, numerical values are assigned to each primary interpretation. Minor discrepancies are defined as differences less than +/-2.0. Major discrepancies are defined as differences greater than or equal to +/-2.0. For the entire laboratory, the overall percentage of concordant cases was consistently above 80% for each of the 6 mo analysis. Regarding the monthly discrepancies, the proportion of minor discrepancies ranged from 11.09% to 15.44% and the proportion of major discrepancies ranged from 1.40% to 3.56%. The frequency distribution of discrepancies by degree approximates a normal (Gaussian) curve and serves as baseline information that may be used for comparison when there are changes in practice or personnel. The CTs attend slide review sessions conducted by the general supervisor for discussion of cases with major discrepancies. The discrepancy data from individual CTs are useful in counseling and recommending areas for improvement. As the CT and pathologist work cooperatively and in tandem, our system allows for a mechanism by which the expectations and needs of pathologist are communicated to the CT more effectively. We believe our process is a fundamental step in improving CT performance in Nongynecologic cytopathology and keeps the CT informed of complexities of nongynecologic cytopathology.     

Diagnostic value of cytopathology in Barrett esophagus and associated carcinoma

Barrett esophagus, the columnar-lined distal esophagus acquired as a consequence of chronic gastroesophageal reflux, is associated with the development of columnar epithelial dysplasia and esophageal adenocarcinoma. To determine the efficacy of cytopathology in identifying Barrett esophagus and related neoplasia, observations were compared on 150 esophageal cytology samples with concurrent endoscopic biopsy specimens. Sixty-six specimens that contained benign columnar epithelium in either cytologic or biopsy material were identified. Distinctive-type Barrett mucosa with incomplete intestinalization, considered diagnostic of Barrett esophagus, was found in 34 of 66 cases (52%) and was present only in cytologic material in 11 cases. Twenty-two specimens contained cardiac-type mucosa (present only in cytology in ten cases), a finding of uncertain significance due to lack of localization of the sample with respect to the gastroesophageal junction. Fundic-type mucosa was not observed in any specimen. Two cases of distinctive-type Barrett mucosa with columnar epithelial dysplasia were identified in both biopsy and cytology specimens. Among eight Barrett-associated carcinomas (seven adenocarcinomas and one squamous), cytologic material was diagnostic for malignancy in seven and highly suspicious in one. It was concluded that cytopathologic studies are a useful adjunct to biopsy histopathology in the diagnosis of Barrett esophagus and associated carcinoma. The role of cytopathology in the diagnosis of Barrett-related columnar epithelial dysplasia requires further study, and at present a cautious approach with biopsy confirmation is recommended.     

Advanced imaging technology applications in cytology

Novel techniques have been developed to image cells at cellular and subcellular levels. They allow images to be analyzed with ultra‐high resolution, in 2D and/or 3D. Several of these tools have been tested on cytology specimens demonstrating emerging applications that are likely to change the field of cytopathology. This review covers several of these advanced imaging methods. The use of optical coherence tomography to perform optical biopsies during endoscopic ultrasound procedures or visualize cells within effusion samples is discussed. The potential for quantitative phase microscopy to accurately screen Pap test slides or resolve indeterminate diagnoses in urine cytology is reviewed. The article also examines the application of 3D cytology using LuCED for lung cancer detection in sputum samples and the feasibility of imaging flow and mass cytometry to measure multiple biomarkers at the single cell level. Although these novel technologies have great potential, further research is necessary to validate their routine use in cytopathology practice.     

Reproducibility study of cervical cytopathology in mexico: A need for regulation and professional accreditation

Due to the high rate of false negative results in diagnosis of cervical cytopathology, in many countries its practice has been transformed through the application of several interventions aimed at medical regulation to improve diagnostic accuracy. Diagnostic reproducibility of gynecological cytopathology was evaluated in a series of 20 cytology specimens [Papanicolaou (Pap)] and 20 cervical biopsy (CB) studies in different clinical stages, during 1994. The observation unit consisted of 30 pathologists who observed 2 groups of 20 Pap and 20 CB specimens. The standard was a cytopathologist certified by the Pathological Anatomy Council of Mexico. Intraclass reproducibility in gynecological cytopathology is low in Mexico. In a group analysis, concordance increased as clinical status of the cervical lesion increased. For moderate dysplasia, concordance in Pap was kappa = 0.04, compared to 0.23 in CB. Concordance of diagnosis of invasive cancer was 0.29 for Pap and 0.64 for CB. Using weighted kappa at the individual level for all possible diagnoses, concordance varied from 0.29 to 0.59 for Pap, and 0.42 to 0.65 for CB. The problem of reproducibility in cervical cytopathology in Mexico emphasizes the need for continuing education, uniform diagnostic criteria, and the advantages of a single operational classification—possibly the Bethesda System—since current classification systems are obsolete. Diagn. Cytopathol. 17:20–24, 1997. © 1997 Wiley-Liss, Inc.     

Diagnostic pitfalls in infectious disease pathology

Numerous images observed in histopathology and cytopathology can mimic pathogens such as mycotic agents, parasites (protozoa or helminthes), bacteria or virus. An error of diagnosis may provoke the administration of a non efficient treatment which can also be toxic. The present review describes these false pathogens which can correspond to exogenous or endogenous agents. Basic morphological analysis of mycotic agents, parasites, bacteria and cytopathogenic viral effects are successively presented. Then, the main diagnostic pitfalls which can be observed during these infections are exposed. Finally, diagnostic problems occurring in case of contamination are rapidly cited.     

Validation in the cytopathology laboratory: its time has come

The cytology laboratory has traditionally performed a relatively small variety of tests. The testing processes employed were generally manually performed and included preparation of glass slide materials, staining of these slides, coverslipping, and microscopic evaluation of the cellular material. Instrumentation in the cytology laboratory was very limited and included a centrifuge, membrane filtration system, and possibly an automated staining machine. If instruments were added, for example, a liquid based preparation device or an automated coverslipping device; the instruments were rarely checked to assure they were operating properly before implementation into clinical practice. In addition, little documentation was maintained with regards to the instrument performance evaluation process. Increasing automation and expansion of testing options have changed how cytopathology is practiced. There are many new devices employed for the preparation of specimens, staining and coverslipping of slides, and evaluation of cellular material. The increasing use of molecular testing methods in cytopathology further adds to the changing landscape of cytopathology. New instrumentation and testing methods are routinely being introduced and the cytopathology laboratory must assure that the testing performed is accurate and consistent. Cytopathology laboratory professionals need to appreciate the value of validation of the tests we perform and the instruments we use in order to best serve the patient. Our clinical laboratory colleagues have traditionally performed validation on both instruments and test methods before using them for clinical testing. If cytopathology wants to perform the complex testing being introduced and effectively utilize new instrumentation, we need to understand the value of validation and how we apply validation to our laboratory practice.     

Urine cytopathology: Challenges, pitfalls, and mimics

Urine cytopathology is a useful and noninvasive tool in the diagnosis and follow‐up of urothelial neoplasia, which remains complementary to emerging molecular tests. These specimens may be challenging and there are numerous mimics and diagnostic pitfalls with which to contend. This review discusses these various entities and includes consideration of ancillary tests that may be useful in the diagnostic procedure. Diagn. Cytopathol. 2012. © 2011 Wiley Periodicals, Inc.     

Telomerase and human papillomavirus as diagnostic adjuncts for cervical dysplasia and carcinoma

Telomerase and human papillomavirus (HPV) DNA were evaluated as potential markers of high-grade dysplasia in cervical cytological specimens. Cytology specimens were collected from patients at the time of colposcopic evaluation for management of a previous abnormal cytology test result. Telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP), and HPV DNA was detected by polymerase chain reaction with L1 consensus-sequence primers and filter hybridization genotyping. Telomerase was detected in 8 of 97 (8.2%) cases with normal cytology or benign cellular changes, in 7 of 98 (7.1%) cases of atypical squamous cells of undetermined significance (ASCUS), in 3 of 95 (3.2%) cases of low-grade squamous intraepithelial lesion (LSIL), and in 17 of 48 (35.4%) cases with high-grade squamous intraepithelial lesion (HSIL). High-risk HPVs were detected in 23 of 97 (23.7%) cases with normal/reactive cellular changes (RCC) cytology, in 28 of 98 (28.6%) cases of ASCUS, in 69 of 95 (72.6%) cases of LSIL, and in 35 of 48 (72.9%) cases of HSIL. Telomerase expression did not correlate with the detection of high-risk HPVs in any cytological diagnostic categories. Telomerase and HPV test results of cytological specimens were correlated with the histological diagnoses of concurrent cervical biopsy specimens. Telomerase showed a sensitivity of 29.9% and a specificity of 94.0% for biopsy-confirmed cervical intraepithelial neoplasia (CIN) II/III. In contrast, high-risk HPVs were detected in 70.1% of cases with underlying CIN II/III, with a specificity of 62.5%. A relatively high proportion of normal/RCC or ASCUS cases with telomerase-positive test results had underlying high-grade dysplasia on cervical biopsy. Thus, technical and practical limitations of the TRAP assay in cervical cytology specimens limit the practical application of telomerase as a diagnostic adjunct in cervical cytopathology.     

Intraoperative and on-site cytopathology consultation: utilization,limitations, and value

The intraoperative and on-site cytopathology can be successfully performed with a number of smear preparations. Specimen concentration technique is the preferred method. The Romanowsky stain such as Diff Quik used in our laboratory is economical, convenient, reproducible, and quick. This can be at times combined with Ultrafast pap or other rapid stains, which provide good nuclear details. Such evaluations are most valuable in the staging of epithelial tumors and primary diagnosis of a number of central nervous system lesions. On-site and intraoperative diagnoses help triage the specimens for additional studies. This reduces the turn around time and makes the procedure cost-effective and beneficial to the patient. An awareness of the "normal" adjacent structures and familiarity with the cytomorphology of the specimens in comparison to the exfoliative specimens is critical in developing a cytopathology service with high degree of sensitivity and specificity.     

The diagnostic and research applications of flow cytometry in cytopathology

Flow cytometry (FCM) is an established ancillary technique applied to the diagnosis of hematological malignancies and for measurement of DNA content. In recent years, the number of fluorochrome-conjugated antibodies available for immunofluorescence and FCM has greatly increased, making it possible to evaluate this technique in other diagnostic contexts, as well as to study a range of biological processes. Serous effusions are optimal for studies using FCM as they consist of viable cells in suspension. Molecules that have been studied for their expression and clinical role in effusions in recent years participate in central cellular functions, including adhesion, proliferation, cellular metabolism, and apoptosis, as well as intracellular signaling. FCM can further be applied to quantitative analysis of molecules related to chemotherapy response, which, together with apoptosis, may represent an important tool for evaluating treatment response and prognosis in advanced and/or recurrent cancer. As targeted therapy assumes an ever-growing role in the treatment of metastatic cancer, the ability to study living cells in effusions or fine-needle aspirates is becoming more important. This article reviews the currently available data in this area of cytopathology.     

Whole slide imaging in cytopathology education

Rapid advances are occurring in the field of cytopathology education and training. Web-based cytopathology educational resources and whole slide imaging (WSI) have revolutionized cytopathology education and helped to centralize the cytopathology resources enabling simultaneous delivery of interactive cytopathology educational programs to a wide range of students and learners nationally and across the globe. WSI is playing a central role in digital pathology and are being utilized as an educational tool in many areas of pathology and cytopathology. This is mainly due to their easy access from anywhere and anytime, with no need for replication of glass slides or a big concern about the issue of protection of patient privacy. Today, WSI is used in a variety of educational settings, as a substitute to multi-headed microscopic sessions, multisite conferences, cytopathology web pages, self-assessment in cytology, cytology proficiency testing, virtual atlases, and very recently in scientific publications.     

Cytopathology of lung cancer: moving from morphology to molecular

The recent discovery and availability of new targeted therapies for lung cancer has presented new challenges to pathologists. Since many lung cancers are diagnosed by aspiration or exfoliative cytology specimens in the primary or metastatic setting, these new therapies have had an impact on the practice of respiratory cytopathology. In particular, accurate subclassification of non-small cell carcinomas and acquisition of sufficient material for molecular studies is crucial. Balancing the need of adequate material for accurate diagnosis with the demands of clinicians to do more with less tissue has been a challenge and continues to impact the way lung cancers are approached in cytopathology. This review focuses on the changes and impact of the molecular era on the diagnosis of lung cancer in cytopathology.     

Molekularpathologische Diagnostik in der Zytopathologie des nichtkleinzelligen Lungenkarzinoms

Background

Personalized medicine is becoming standard for the treatment of non-small-cell lung cancer. For example, patients with activating EGFR mutations or EML4-ALK translocations largely benefit from targeted therapies with tyrosine kinase inhibitors with better response rates and progression-free survival compared to standard chemotherapy regimens. However, the application of the respective molecular biomarker analyses requires great expertise in the handling of different cell and tissue specimens. A major challenge for reliable analyses is the usually low amount of tumor material. There are currently relatively few standardized and evidence-based guidelines for the processing and analysis of respective specimens as well as for interpretation of the test results.

Materials and methods

To establish a basis for standardized predictive cytopathological analyses, different material processing approaches and molecular pathological tests are discussed, and novel concepts and strategies are lined out in order to improve the quality and reliability of the respective diagnostic procedures.

Results and discussion

Predictive analyses of cytological specimens can be reliably performed using smears, cytospins or cell blocks; there is no need for histological specimens. The diagnostic work-up of cytological probes should be performed as carefully as possible in order to save further tumor material for subsequent predictive analyses. With standardized and reliable procedures at hand cytopathology is an important contribution to the multidisciplinary, complex care, and treatment of lung cancer patients.     

Intra-operative cytopathology: techniques, indications, and limitations

Intraoperative cytopathology is faster, less labor intensive, yields clearer cellular details and can be as accurate as frozen section in the hands of pathologists experienced in the interpretation of cytological preparations. This procedure is particularly valuable for examining small specimens, multiple samples that need to be examined rapidly, and when superior cytological details are required. Nonetheless its use seems to be relatively limited. In this article, we review the general requirements for intraoperative cytology and also detail its value, as well as its limitations and pitfalls.     

Cytologic-histologic correlation of nongynecologic cytopathology cases: separation of determinate from indeterminate cytologic diagnoses for analysis and monitoring of laboratory performance

Much of the literature on the quality-assurance aspect of cytologic-histologic correlation (CHC) has focused on gynecologic cytology. For nongynecologic cytopathology, the process is complicated by the use of determinate (positive for malignant cells, negative for malignant cells) and indeterminate (atypical, suspicious, or follicular lesion) diagnostic categories. Here, we illustrate our routine methodology for analyzing CHC data on nongynecologic cytopathology cases by separating determinate from indeterminate cases. A focused list of determinate and indeterminate cytopathology cases with surgical pathology correlation is generated each week. The determinate cases are ascertained as true positive (TP), true negative (TN), false positive (FP), or false negative (TN). The discrepant cases (FP and FN) are investigated to determine the cause (sampling, interpretation, or screening). For indeterminate cases, the surgical pathology outcome (benign, malignant) and suitability of the cytopathology category utilized are reviewed. For the focused period of 4 mo, sensitivity was 70% and specificity was 100%. The most common reason for false-negative diagnoses was a sampling problem in the cytologic specimen; there were no false-positive diagnoses. Malignant outcomes for follicular lesion, atypical, and suspicious diagnoses were 29%, 40%, and 76%, respectively. Data derived from regularly performed CHC are useful in reviewing the diagnostic performance of the laboratory.     

Discrimination of cell types in primary transitional cell carcinoma by monoclonal anti-cytokeratin antibodies.

The morphologic discrimination between low and high grade malignant tumor cells arising in the urinary bladder is a major diagnostic problem in cytopathology. Using immunochemical peroxidase staining of cytokeratins (CKs) of human bladder exfoliative cytology specimens, we have been able to discriminate between transitional cell carcinoma cells, atypical cells and normal bladder cells. Commercially available monoclonal antibodies used in this study were: anti-CK 13 (Sigma K8.12), anti-CK 5, 7 and 8 (Sigma K8.13), anti-CK 19 (Sigma K4.62), and anti-CK 18 (Transformation Res. 1091). When normal bladder cells are stained with these antibodies, CK 18 is specific for superficial cells; CK 13 is specific for the basal type cells and CKs 5, 7, 8 and 19 are expressed by all urothelial cell types. Four cases diagnosed by cytopathological criteria as 'atypical' and 7 diagnosed as 'positive' (various grades) were used in this study. Cytologic diagnosis was confirmed by histopathology in 7 cases. Tissue was not available for histopathology in 4 cases. Malignant cells with a 'basal' or 'immature' phenotype preferentially stained with CK 13 and were associated with increased metastatic or malignant potential. Patients with higher grade tumors had more cells positive for CK 13 than did patients with atypical or lower grade malignancies. Patients with well-differentiated, low grade tumors predominantly shed cells that expressed CK 18 and CK 19. High grade, invasive bladder lesions were characterized by many cells expressing CK 13, and fewer cells expressing CK 19. The combination of diagnosis by morphologic criteria on Papanicolaou-stained specimens with immunochemical characterization of the same cells for CKs facilitate an accurate diagnosis of bladder lesions.     

Locus coeruleus neurofibrillary degeneration in aging, mild cognitive impairment and early Alzheimer's disease

Neurofibrillary degeneration in the nucleus basalis and a loss of its cortical cholinergic projections are prominent components of the neuropathology in Alzheimer's disease (AD). The AD brain is also associated with a degeneration of the noradrenergic projections arising from the nucleus locus coeruleus (LC), but the time course of this lesion is poorly understood. To determine whether the LC displays neurofibrillary abnormalities early in the course of events leading to AD, we examined tissue specimens from seven cognitively normal controls and five subjects at the stages of mild cognitively impairment (MCI) or early AD. Tyrosine hydroxylase immunochemistry was used as a marker of LC neurons while AT8 immunolabeling visualized abnormal tau associated with neurofibrillary tangles and their precursors. Thioflavine-S was used as a marker for fully developed tangles. We found that AT8-positive labeling and thioflavine-S positive tangles were present in both groups of specimens. However, the percentage of neurons containing each of these markers was significantly higher in the cognitively impaired group. The MMSE scores displayed a negative correlation with both markers of cytopathology. These results indicate that cytopathology in the LC is an early event in the age-MCI-AD continuum and that it may be listed among the numerous factors that mediate the emergence of the cognitive changes leading to dementia.