Immunocytochemistry on cytologic specimens of limited quantity

Some cytologic specimens may be limited in quantity, and this may hamper or preclude the performance of immunocytochemistry (ICC) in cases where more than one antibody (ab) is required by ICC to arrive at a definitive diagnosis. There is very little information in the cytology literature regarding the use of ICC for specimens that are limited in quantity. In this study, we describe a method, derived from the principles of double immunolabelling, whereby more than one ab test can be repeatedly used on the same Papanicolaou stained slide. Multiple cytologic scrape preparations fixed in 95% ethanol were obtained from fresh surgical specimens including carcinomas of the breast, endometrium, stomach, ovary and colon. Nonneoplastic tissues included tonsil (2), lymph node (2) and myometrium. Papanicolaou stained slides or unstained slides were subjected to two sequential ICC procedures, the first in which the ab was known to be nonreactive with the cells (insulin, glucagon, or somatostatin) and the second in which the ab was known to be positive in the cells. Positive controls for the known positive abs included a single-step ICC procedure as well as the tissue section. The test abs included CAM 5.2, AE1/3, K903, LCA, L26, UCHL-1, s-100, mCEA, GCDFP-15, vimentin, muscle specific actin and desmin. Identical two-step ab procedures were carried out on the tissues from the same surgical specimens. For Papanicolaou stained cytologic specimens, abs were reactive and gave excellent results for the repeat second-step ICC method. There was no false positive or false negative staining. This “repeat ICC” method also gave excellent results on the tissue sections. Immunocytochemistry can be performed more than once on the very same cytologic specimen if the initial ICC antibody attempt is negative. This method may be especially useful in situations where more than one antibody is needed on a very limited cytologic sample size. Diagn. Cytopathol. 1998;18:166–169. © 1998 Wiley-Liss, Inc.     

Rapid cytologic examination of surgical specimens: a valuable technique in the surgical pathology laboratory

Rapid cytologic examination of resected tissue has been shown to be a useful adjunct to the use of frozen sections, as well as an efficient method for learning to interpret clinical fine-needle aspirates. Cytologic preparations afford better cytologic detail and sampling capability than frozen sections, and use of the two procedures together is advocated. Methods are described, diagnostic criteria for selected tissues are reviewed, and specific examples of the usefulness of imprints with examples of diagnostic problems are presented.     

Distinguishing tall cell variant of papillary thyroid carcinoma from usual variant of papillary thyroid carcinoma in cytologic specimens

Tall cell variant (TCV) is an aggressive form of papillary thyroid carcinoma (PTC), usually associated with higher local recurrence and distant metastasis. Some authors have suggested that TCV can be effectively diagnosed on thyroid fine-needle aspiration (FNA); this diagnosis may help clinicians plan a more effective treatment regimen. The objective of this study was to compare the FNA specimens of TCV with those of usual variant of PTC (UV-PTC) and to define a set of distinguishing cytologic features. Thirty FNA specimens of histologically proven TCV were compared with 32 FNA specimens of histologically proven UV-PTC. All specimens were evaluated for the following features: papillary groups (PG), elongated/tall cells (EL/TC), oncocytic cytoplasm (OC), distinct cell borders (DCB), prominent central nucleoli (PCN), intranuclear grooves (NG), and intranuclear inclusions (NI). These features were semiquantitatively measured on a sliding scale of 0-4 in both air-dried Diff-Quik-stained and ethanol-fixed Papanicolaou-stained preparations. TCV showed distinctive cytologic features, which can distinguish them from UV-PTC. These included EL/TC, OC, and DCB and were also found to be statistically significant (P < 0.0001). No significant differences were noted for PG and NG. The NIs in TCV cases were qualitatively different than those in UV-PTC. In TCV there were multiple inclusions within the same nucleus imparting a "soap bubble appearance" to the nucleus. This feature was seen in almost all cases of TCV and was rarely seen in usual PTC. On the basis of the above-mentioned cytologic features, TCV can be distinguished from usual PTC in FNA specimens.     

Tumor genes and their proteins in cytologic and surgical specimens: Relevance and detection systems

Oncogenesis is the consequence of a series of genetic alterations that allow unrestrained cellular growth, tissue invasion, and eventual metastases. Tumor-related genes can be classified into functional categories. Proto-oncogenes/oncogenes have a stimulatory role in cell growth, and the inactivation of cancer-suppressor genes/antioncogenes results in the loss of cell cycle regulation. More recently, three other groups of tumor-related genes have been recognized. They include the antiapoptosis genes which protect from programmed cell death, the antimetastasis genes, and multidrug resistance genes. Besides aiding in tumor diagnosis, the detection of such tumor-associated genes and their products allows the identification of individuals with an inherited predisposition to neoplastic growths, and the overexpression of many of these oncogene products has been shown to be a potential marker of tumor behavior and a predictor of treatment outcome and response. The ability to utilize DNA and RNA probes for nucleic acid hybridization and polymerase chain reaction procedures in cell and tissue preparations of solid tumors and lymphoid proliferations expands and complements the information provided by immunohistochemical techniques. These probes allow direct visualization and correlation of specific genes and their protein products with cytomorphologic features, and form a powerful addition to the armamentarium of the cytopathologist and surgical pathologist. © 1995 Wiley-Liss, Inc.     

Evaluation of apoptosis in cytologic specimens

A hallmark of neoplasia is dysregulated apoptosis, programmed cell death. Apoptosis is crucial for normal tissue homeostasis. Dysregulation of apoptotic pathways leads to reduced cytocidal responses to chemotherapeutic drugs or radiation and is a frequent contributor to therapeutic resistance in cancer. The literature pertaining to detection of apoptotic pathway constituents in cytologic specimens is reviewed herein. Virtually all methods for detecting apoptosis, including classic cytomorphologic evaluation, TUNEL assay, immunocytochemistry, and gene sequence analysis, may be applied to cytologic samples as well as tissue. Components of both intrinsic and extrinsic apoptotic pathways have been studied, including many reports examining p53 and bcl‐2, as well as studies of caspase inhibitory proteins XIAP and survivin, death receptors and ligands such as Fas, Fas‐ligand, and TRAIL. p53 undergoes oncogenic alteration more than any other protein; its immunocytochemical detection almost always connotes loss of its physiologic role as an inducer of apoptosis in response to a damaged genome. Several reports establish cytologic sampling as being as useful as tissue sampling. In one respect cytologic sampling is superior to tissue sampling in particular, by allowing clinicians to repeat sampling of the same tumor before and after administration of therapy; a number of reports use this approach to attempt to predict tumor response by assaying the effect of chemotherapy on the induction of apoptosis. Diagn. Cytopathol. 2010;38:685–697. © 2010 Wiley‐Liss, Inc.     

Matrix crystals in cytologic urine specimens.

Three cytologic urine specimens from separate patients seen over a period of 3 mo were prepared by the Papanicolaou method. They contained crystals (uric acid type in two and magnesium ammonium phosphate in one) that incorporated variable amounts of organic (mucoprotein) matrix; many appeared by light microscopy to be made exclusively of matrix. Scanning electron microscopy performed in one of the specimens containing uric acid crystals suggested that the matrix forms were in progressive stages of mineralization. These cases, plus a similar one reported recently by us, demonstrate that the detection by urine cytology of organic matrix incorporated in the structure of urinary crystals is not rare and that the Papanicolaou staining method facilitates such detection.     

Immunophenotyping of cytologic specimens by flow cytometry

Immunophenotyping by flow cytometry is well established as an ancillary technique in the diagnosis of hematopoietic neoplasms. However, flow cytometry is rarely performed on cytologic specimens because most cytologists are more comfortable with direct microscopy and believe that there is inadequate cellularity for analysis. Paradoxically, cytologic material is usually cell suspensions making it ideal for flow cytometry. In order to evaluate the usefulness of immunophenotyping cytologic specimens by flow cytometry, we retrospectively reviewed all cytologic specimens submitted to our flow cytometry unit from 1988 to 1991. Thirty-one cerebrospinal fluid specimens were analyzed. There were inadequate cells for analysis in 15 cases. Five showed a monoclonal proliferation; 11 were nondiagnostic. A range (r) of one to six cell surface markers were performed. Thirty-two body cavity fluids were analyzed: 7 peritoneal, 19 pleural, 2 pericardial, and 4 bronchoalveolar lavage. There were cells to analyze in all cases. Seven had a monoclonal proliferation; 25 were nondiagnostic (r = 4-21 markers performed). One hundred eighteen fine needle aspirates (FNA) were reviewed; 58 FNA were radiologically guided, 60 were superficial lesions. There were inadequate cells for analysis in two cases. Sixty-one demonstrated a monoclonal proliferation; 55 were nondiagnostic (r = 1-22 markers performed). We conclude that immunophenotyping by flow cytometry is of limited value for cerebrospinal fluid analysis and that knowledge of previous immunophenotyping studies is essential for correct analysis; analysis of body cavity fluids is easily performed but less often demonstrates a monoclonal proliferation. Immunophenotyping by flow cytometry is a valuable adjunctive technique for FNA and yields adequate cells for analysis.     

Morphologic features of endometriosis in various types of cytologic specimens

Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. This study evaluates the cytomorphologic features of endometriosis in various cytologic specimen types [fine‐needle aspiration (FNA), effusion cytology (EF), touch imprint (ToP), and cervical smear (PAP)], and assesses the key elements helpful in recognizing this lesion. A total of 18 cases (8 FNA, 4 EF, 5 ToP, and 1 PAP) of cytologically diagnosed and histologically/clinically confirmed endometriosis diagnosed between 1988 and 2006 comprises the material for this study. The morphologic features evaluated of the three components included: cellularity, presence of sheets of glandular cells, three‐dimensional (3D) glandular clusters, tubular structures, single cells, syncytial groups of stromal cells, stromal cells entrapped within basement membrane (BM)‐like material, cytologic atypia, presence of mitotic figures, and hemosiderin‐laden histiocytes. Endometrial glands, stroma, and hemosiderin‐laden histiocytes were all identified in 14/18 (77.8%) cases. FNA specimens were more cellular than that of both EF and ToP specimens. Tubular structures, 3D glandular clusters, stromal cells entrapped in BM and syncytial stromal groups were more common in FNAs, and ToPs compared with the EFs. The ratio of the endometrial glandular and stromal cells was similar in all specimen types. Atypia and mitotic figures were rarely encountered. Diagnosis of endometriosis could be made independently on either smears/ThinPrep^? slides or on cell blocks in all cases where these preparations were available. On follow up, none of the patients developed malignancy. Endometriosis can be reliably and safely diagnosed in various cytologic materials. Cytologic atypia is uncommon. Components of endometriosis could show minor morphologic alterations in different specimen types. Diagn. Cytopathol. 2013;41:936–942. © 2013 Wiley Periodicals, Inc.     

Cytokeratins 20 and 7 in the differential diagnosis of metastatic carcinoma in cytologic specimens

A series of 33 tumor cases (13 fine-needle aspirates and 20 effusion specimens) were evaluated for the expression of two cytokeratin (CK) subtypes; CK 20, expressed primarily in tumors of the GI tract, mucinous ovarian tumors and transitional cell carcinomas, and CK 7, found chiefly in non-GI tract adenocarcinomas, including breast and lung. CK 20 expression was demonstrated immunocytochemically in seven of seven metastatic colon and two of three metastatic transitional cell carcinomas tested. CK 20 was absent in all metastatic carcinomas of breast, ovary, lung, and uterus examined (23 cases). Anti-CK 7 stains were negative in four of six metastatic colonic carcinomas, with equivocal results in the remaining two cases. Metastatic lung, breast, and ovarian carcinomas were strongly positive for CK 7. This study demonstrates that the combined use of anti-CK 20 and anti-CK 7 antibodies is highly sensitive and specific for metastatic colonic adenocarcinoma in cytologic material and thus could play an important role in distinguishing this entity from other common primary carcinomas. Diagn. Cytopathol. 16:132–136, 1997. © 1997 Wiley-Liss, Inc.     

Dipeptidyl aminopeptidase IV in the cytologic diagnosis of thyroid carcinoma

Recently, the demonstration of DAP IV activity in thyroid cells aspirates has been proposed as an useful tool for the diagnosis of malignancy. We have studied the enzymatic activity of DAP IV, using the modified method of Lodja, in a series of 336 selected aspirates of the thyroid gland with the following cytologic diagnosis: 236 nodular hyperplasias, 60 follicular proliferations, eight Hashimoto's thyroiditis, eight Hürthle-cell proliferations, 20 papillary carcinomas, two anaplastic carcinomas, and two medullary carcinomas. The results were subjectively evaluated on the basis of staining intensity and extension in a minimum of 200 cells. Strong-to-moderate enzymatic activity with an extension of more than 40% of the cells were exclusively seen in follicular-cell derived carcinomas (papillary carcinoma, Hürthle-cell carcinoma, and follicular carcinoma). Medullary carcinoma, anaplastic carcinoma, and benign conditions were negative or weakly stained. Cytohistologic correlation in 88 patients operated on showed the following results: 26 nodular hyperplasia (18 nodular hyperplasia and eight follicular adenomas), 36 follicular proliferation (24 nodular hyperplasia, six, adenomas, three papillary carcinomas, three follicular carcinomas), two Hürthle-cell proliferation (one Hürthle-cell adenoma and one Hürthle-cell carcinoma), 20 papillary carcinomas, two medullary carcinomas, and two anaplastic carcinomas. DAP IV staining was moderate to strong and extensive in all malignant tumors initially diagnosed as follicular or Hürthle-cell proliferations. We conclude that DAP IV activity is present in malignant differentiated thyroid tumors of follicular cells (papillary carcinoma, follicular carcinoma, Hürthle-cell carcinoma), but it is identified neither in medullary carcinoma nor in anaplastic carcinoma. Therefore, its usefulness is restricted to the diagnosis of follicular-cell malignancies. Diagn. Cytopathol. 1998;19:4–8. © 1998 Wiley-Liss, Inc.     

Immunocytochemistry in thyroid cytology and its multiple roles: a systematic review

The limits of morphological analysis, mostly encountered in the diagnosis of “follicular thyroid neoplasms (FN)”, were clearly reported in many papers. For this reason, the application of ancillary techniques, including immunocytochemistry (ICC) and molecular testing, contributes to a better definition of the risk of malignancy (ROM) and management of thyroid nodules (TN). This review discusses the diagnostic and prognostic role of the most relevant immunomarkers in TN, including HBME-1, Galectin-3, CD56, and VE1-BRAF among the others. Furthermore, we also analyzed the growing interest for the so-called next-generation immunomarkers, which represent one of the new frontiers for cancer scientific research. According to the literature, the diagnostic and prognostic relevance of their performance is able to help the morphological evaluation, especially for the indeterminate categories and controversial diagnoses. In fact, even though ancillary techniques cannot substitute the relevance of morphological evaluation of thyroid samples, they might represent a valid adjunct for those cases in which controversial features are not able to lead to a univocal diagnosis. Herein the review of literature about each of the immunomarkers in thyroid nodules.     

Expression of estrogen and progesterone receptors in cytologic specimens using various fixatives

Estrogen and progesterone receptor reactivity may be useful in identifying possible primary sites of metastatic disease or directing therapy in tumors of the female genital tract, including breast, ovary, and endometrium. Various methods have been described for the immunocytochemical evaluation of estrogen receptor (ER) and progesterone receptor (PR) status of cytologic specimens but our results have been variable. We evaluated the effectiveness of various fixatives [cytospin collection fluid, Shandon, Pittsburgh, PA (SH); ethanol (ETH); and formalin (FOR)] for fixation of smears (SM) and cell block (CB) material. The percentage and intensity of tumor nuclei of SM, CB, and tissue sections (TS) stained for ER and PR by the avidin-biotin-peroxidase complex technique were compared. Samples were considered ER or PR positive when ≥20% of tumor nuclei were stained. The sensitivity of ER analysis of SMs and CBs in each fixative compared to formalin-fixed paraffin-embedded tissue sections were as follows, SM (SH) 88%, SM (ETH) 14%, CB (SH) 58%, CB (ETH) 43%, and CB (FOR) 70%. The sensitivity of PR determination on SMs and CBs was SM (SH) 71%, SM (ETH) 6.0%, CB (SH) 25%, CB (ETH) 33%, CB (FOR) 80%. These findings indicate that of the fixatives evaluated for ER analysis SMs fixed in SH provided the best results. For PR evaluation, CBs fixed in FOR gave the best results. Diagn Cytopathol 1996;15:78–83. © 1996 Wiley-Liss, Inc.     

Preoperative cytologic diagnosis of medullary carcinoma of the thyroid

The Authors emphasize the importance of preoperative aspiration cytology in the diagnosis of 5 medullary types out of 58 thyroid carcinomas observed in the Institute of Pathology of the Università Cattolica del S. Cuore of Rome in the period 1983-88. All the cases were diagnosed preoperatively by mean of a fine-needle aspiration cytological specimen; the case where morphological diagnosis was doubtful were studied with immunohistochemical techniques for calcitonin and CEA. Out of all cases, they report one case of multiple endocrine adenomatosis type II with medullary thyroid carcinoma and pheochromocytoma and another one with cervical node metastases without a clinically detectable thyroid swelling. Therefore, the authors propose the fine-needle aspiration cytology as a simple and important mean in the preoperative diagnostic procedure in case of medullary thyroid carcinoma.     

Challenges for the pathologist in the interpretation of laparoscopic hysterectomy and cytologic specimens in endometrial carcinoma

The introduction of new and improved surgical techniques for treatment of endometrial carcinoma has been shown to be associated with histological artifacts making interpretation and diagnosis a challenge for the pathologist with an impact on clinical staging. In this review, challenging artifacts associated with the use of manipulated laparoscopic hysterectomies for endometrial carcinoma are discussed with a review of the literature. Topics discussed include the artifacts of vascular pseudoinvasion; endomyometrial cleft displacement, disruption of endometrial lining, nuclear crush artefact and positive peritoneal cytology. The pathologist should be aware of these artifacts in order to prevent diagnostic errors and unwarranted therapy for some patients.     

Update on the cytologic features of papillary thyroid carcinoma variants

Papillary thyroid cancer (PTC), which accounts for 85–90% of all thyroid cancers, is generally an indolent tumor with long term survival rates >95%. A reliable definitive diagnosis of PTC is usually straightforward in fine needle aspirates of conventional PTC whenever the characteristic papillary and/or flat honeycomb sheet‐like architecture and the typical nuclear features of chromatin pallor, nuclear enlargement, crowding, grooves and pseudoinclusions are encountered. Conventional PTC, however, has diminished in relative frequency as compared to PTC variants, especially the noninvasive follicular variant of PTC, an indolent tumor which has recently been reclassified as “noninvasive follicular thyroid neoplasm with papillary‐like nuclear features” (NIFTP). These PTC variants are characterized by various architecture, cell type and shape, and stromal features, some of which can be recognized cytologically. Awareness of the cytomorphological spectrum and of the characteristic cytological features of these PTC variants is important to avoid diagnostic pitfalls. In this article, we review the different variants of PTC, including their cytomorphologic features, differential diagnosis, and salient molecular features. Diagn. Cytopathol. 2017;45:714–730. © 2017 Wiley Periodicals, Inc.