Cytokeratin Immunocytochemistry Research Articles

Usefulness and Limitations of Cryopreservation for Immunocytochemical Staining of Canine Cytological Specimens for Detection of Cytokeratin and Vimentin.

Immunocytochemistry is an advanced diagnostic tool for identifying the origin of tumor cells. This study aimed to highlight the usefulness of cryopreserved, air-dried cytological samples in detecting cytokeratin and vimentin. Air-dried cytological smear samples were prepared from a total of 39 resected canine tumors and stored in a medical freezer without fixation. The duration of cryopreservation ranged from 2 to 56 months. The same tumors were processed for routine histopathological examination. Based on the morphological diagnosis, cryopreserved FNA smears from epithelial tumors were stained by enzymatic immunocytochemistry (ICC) for cytokeratin; those from mesenchymal and melanocytic tumors were stained by ICC for vimentin. To ascertain the positivity of tumor cells to the selected markers, tissue paraffin-embedded sections were also stained by immunohistochemistry (IHC) for the same markers. Immunoreactivity for cytokeratin was detected in cryopreserved cytological smears for a maximum of 46 months. Immunoreactivity for vimentin was clearly detected for 33 months. Smears stored at room temperature for 1 week did not show any signals under immunocytochemical examination. Thus, immunocytochemistry for cytokeratin and vimentin can be safely applied to air-dried smears cryopreserved in a freezer for at least 33 months.

Hepatocellular carcinoma risk-stratification based on ASGR1 in circulating epithelial cells for cancer interception.

Purpose: Lack of diagnostic and prognostic biomarkers in hepatocellular carcinoma impedes stratifying patients based on their risk of developing cancer. The aim of this study was to evaluate phenotypic and genetic heterogeneity of circulating epithelial cells (CECs) based on asialoglycoprotein receptor 1 (ASGR1) and miR-122-5p expression as potential diagnostic and prognostic tools in patients with hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Methods: Peripheral blood samples were extracted from LC and HCC patients at different disease stages. CECs were isolated using positive immunomagnetic selection. Genetic and phenotypic characterization was validated by double immunocytochemistry for cytokeratin (CK) and ASGR1 or by in situ hybridization with miR-122-5p and CECs were visualized by confocal microscopy. Results: The presence of CECs increased HCC risk by 2.58-fold, however, this was only significant for patients with previous LC (p = 0.028) and not for those without prior LC (p = 0.23). Furthermore, the number of CECs lacking ASGR1 expression correlated significantly with HCC incidence and absence of miR-122-5p expression (p = 0.014; r = 0.23). Finally, overall survival was significantly greater for patients at earlier cancer stages (p = 0.018), but this difference was only maintained in the group with the presence of CECs (p = 0.021) whereas progression-free survival was influenced by the absence of ASGR1 expression. Conclusion: Identification and characterization of CECs by ASGR1 and/or miR-122-5p expression may be used as a risk-stratification tool in LC patients, as it was shown to be an independent prognostic and risk-stratification marker in LC and early disease stage HCC patients.

Morphometrical, Morphological, and Immunocytochemical Characterization of a Tool for Cytotoxicity Research: 3D Cultures of Breast Cell Lines Grown in Ultra-Low Attachment Plates.

Three-dimensional cell cultures may better mimic avascular tumors. Yet, they still lack characterization and standardization. Therefore, this study aimed to (a) generate multicellular aggregates (MCAs) of four breast cell lines: MCF7, MDA-MB-231, and SKBR3 (tumoral) and MCF12A (non-tumoral) using ultra-low attachment (ULA) plates, (b) detail the methodology used for their formation and analysis, providing technical tips, and (c) characterize the MCAs using morphometry, qualitative cytology (at light and electron microscopy), and quantitative immunocytochemistry (ICC) analysis. Each cell line generated uniform MCAs with structural differences among cell lines: MCF7 and MDA-MB-231 MCAs showed an ellipsoid/discoid shape and compact structure, while MCF12A and SKBR3 MCAs were loose, more flattened, and presented bigger areas. MCF7 MCAs revealed glandular breast differentiation features. ICC showed a random distribution of the proliferating and apoptotic cells throughout the MCAs, not fitting in the traditional spheroid model. ICC for cytokeratin, vimentin, and E-cadherin showed different results according to the cell lines. Estrogen (ER) and progesterone (PR) receptors were positive only in MCF7 and human epidermal growth factor receptor 2 (HER-2) in SKBR3. The presented characterization of the MCAs in non-exposed conditions provided a good baseline to evaluate the cytotoxic effects of potential anticancer compounds.

Minimal Residual Disease in Prostate Cancer.

Detection of minimal residual disease (MRD) in prostate cancer over several decades has greatly informed our understanding of dissemination and recurrence, but has not yet been routinely used in clinical care. Investigators have detected MRD by identification of prostate cancer cells in the bone marrow; termed disseminated tumor cells (DTCs) and blood; termed circulating tumor cells (CTCs). Various techniques including PSA-RT PCR, PSA immunocytochemistry, cytokeratin immunocytochemistry, and immune-magnetic depletion of hematopoietic cells followed by EPCAM based positive selection, have been used. Importantly, detection of DTCs correlates with recurrence. Research into prostate cancer CTCs has intensified recently, but their use in MRD evaluation has been more limited. Investigators are using semi-automated platforms to detect and begin to study prostate cancer CTCs in patients with no evidence of disease. PSA immunocytochemistry also detects CTCs and correlates with recurrence. Emerging technologies have the potential to greatly aid research in this exciting field.

Abstract 819: Transcriptomic profiling of two canine mammary cancer cell lines for translational oncology studies

Abstract Dogs develop spontaneous cancers as humans and shares a variety of features including histology, genomic alterations, molecular targets, biological behavior and response to conventional therapies. Although dogs can be considered good models for translation of new cancer treatments, there is a lack of established preclinical in vitro models of canine cancers. Therefore, our aim here was to establish two canine mammary cancer cell lines and characterize their transcriptomic profile to speed the translation of new cancer treatments from dogs to humans. Mammary cancer samples were obtained from female dogs that underwent surgery. Pieces of the tumors were FFPE for histopathology and the other fragments were washed with PBS, minced and dissociated with hyaluronidase and collagenase. Cells were filtered and put in culture with DMEM-F12, 5%FBS and 1% pen-strep. After 5 passages, FBS was increased to 10%. After passage 15, cells were characterized for doubling time, karyotype, immunocytochemistry for cytokeratin, vimentin and α-smooth muscle actin, matrigel-invasion assay and tumor growth in nude mice. At last, RNA-seq was performed on total RNA extracted with TRIZOL for functional enrichment analysis of differentially expressed (DE) genes from cancer cell lines. From 49 tumor samples, we characterized 2 cell lines named M5 and M25, originally from a comedocarcinoma and a mixed carcinoma, respectively. Both cell cultures showed spindle-shape cells and were positive for cytokeratin, vimentin and α-smooth actin, suggesting myoepithelial origin. The doubling time was 26.0h for M5 and 42.8h for M25 cells. Karyotyping analysis showed moderate aneuploidy for both cell cultures. M25 cell line presented significant higher invasion potential than M5 cells and formed tumors in mice but the latter didn’t. Histopathology of these tumors resembled the original tumor in the dog. An average of 16.2 million paired-end 100 bp reads were sequenced per replicate (three for each cell culture) with an average of 81.1% alignment to the reference genome. A total of 11,260 and 11,476 genes with more than 1 count per million were detected in M5 and M25 cell lines. M5 cell line have 404 and 394 up- and downregulated genes in comparison to M25 cell line, respectively. Gene ontology of DE genes showed the more malignant cell (M25) significantly enriched for pathways related to drug metabolism, ECM-receptor interaction, cell cycle, DNA replication, focal adhesion and cell differentiation. In conclusion, M25 cell line is more malignant than the M5 which is supported by their transcriptome. Thus, it is possible to use these cells as a comparative translational model for drug development especially if one wishes to work with molecular targets as described above. Citation Format: Heidge Fukumasu, Yonara G. Cordeiro, Pedro L. Xavier, Arina L. Rochetti, Pamela A. Alexandre, Claudia C. Mori, Silvio H. Freitas, Ricardo F. Strefezzi. Transcriptomic profiling of two canine mammary cancer cell lines for translational oncology studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 819. doi:10.1158/1538-7445.AM2017-819

Pan-cytokeratin markers for rapid frozen section immunocytochemistry from head and facial Mohs cases of basal cell carcinoma: a comparison and evaluation to determine the marker of choice

ABSTRACTThe application of immunocytochemistry in the field of Mohs micrographic surgery (MMS) is well established. This study evaluates the use of pan-cytokeratins (AE1/AE3, MNF116 and AE1/AE3+PCK26) in the assessment of basal cell carcinoma (BCC) on frozen tissue debulk specimens. Fifty-five cases of BCC, all from head and facial sites, were assessed in the study. In addition to staining all cases for the three cytokeratin antibodies under investigation, sections were also stained with haematoxylin and eosin (H&E) to demonstrate tumour architecture and morphology. All sections for immunocytochemistry were stained on a Roche Ventana BenchMark Ultra automated platform employing a rapid frozen section protocol. Results were assessed based on the intensity of staining of keratinocytes (scale: 0–100%), as well as sensitivity of staining determined by the total percentage of keratinocytes stained within the tissue section. AE1/AE3 demonstrated the most consistent staining both in terms of intensity of staining and sensitivity, with a mean of 99.1% and 99.9%, respectively. AE1/AE3+PCK26 average results indicated scores of 70.6% for intensity and 87.2% for sensitivity, with MNF116 scoring 92.9% for intensity but only 57.3% for sensitivity. The data indicate that AE1/AE3 is the best pan-cytokeratin antibody to use in the assessment of BCC in MMS. The use of cytokeratin immunocytochemistry is justified in morphologically complex cases of BCC, or in cases where dense inflammatory infiltrate surrounding any suspicious cells make identification of small numbers of tumour cells difficult to determine with just an H&E stain. The significant rationale is that cytokeratin staining is a valuable adjunct in the study of tumour cell assessment in cases of MMS for BCC. In addition, the use of anti-AE1/AE3 cytokeratin antibodies provides the most consistent staining results for such cases.

The cytomorphologic spectrum of Wilms tumour on fine needle aspiration: a single institutional experience of 110 cases

To analyse the cytomorphologic spectrum of Wilms tumour (WT) on aspirates, the largest series reported to date. Adequate aspirates from paediatric renal tumours over a period of 17 years were reviewed and selected if subsequent excision showed WT or aspirates were diagnostic for WT and clinical/radiological evidence consistent with that diagnosis. Smears were re-examined for the proportion of components, degree of pleomorphism and mitosis. Of 110 aspirates, smears were triphasic in 44 (40.0%), biphasic (blastema and tubules) in 36 (32.7%) and monophasic (blastema alone) in 30 (27.3%). Stromal predominance was seen in 11 aspirates (10.0%) and five showed rhabdomyoblastic differentiation; all 11 were triphasic. Mean mitotic rate was 9.3/5000 cells (range 4-39/5000). Nuclear atypia not amounting to anaplasia and without atypical mitoses was seen in 15 (13.6%); these presented diagnostic problems. Two aspirates (1.8%) were considered anaplastic (unfavourable), both having atypical mitoses. Criteria similar to histology (i.e. 3-fold or more variation in nuclear size, marked hyperchromasia with bizarre nuclei and atypical mitoses in a biphasic or triphasic aspirate) helped in distinguishing anaplastic WT. Histopathological correlation in 67 cases showed good correlation of blastemal predominance, stromal predominance and anaplastic histology with the corresponding cytology. However, 9/27 (33.3%) triphasic tumours had only blastemal cells on corresponding aspiration because of sampling error. Cytokeratin was positive in 4 of 20 aspirates with blastema alone. Aspirates from WT were triphasic or biphasic in the majority (72.7%), permitting cytological diagnosis, which was improved by cytokeratin immunocytochemistry. Blastemal and stromal predominance on histology correlated well with cytology, but many triphasic tumours showed only blastema on aspiration. Anaplastic WT can be detected on aspirates using criteria similar to histology.

Value of Additional Immunocytochemical Stain for Cytokeratin in the Diagnosis of Leptomeningeal Involvement of Metastatic Carcinoma

Background: The purpose of this study was to describe potential pitfalls in the diagnosis of metastatic adenocarcinoma in cerebrospinal fluid (CSF) and to suggest additional work in association with cytokeratin immunocytochemistry for the proper diagnosis, especially in the specimens with low cellularity. Methods: We collected 267 cytologic specimens of CSF from patients, who were diagnosed over a 9-month period. Each of the individual samples were divided into half the sample size and processed via both, ThinPrep (TP) with Papanicolau stain and cytocentrifugationbased preparation (cytospin, CP) with immunocytochemical stain for cytokeratin. Results: Amongst the 267 cases, 45 cases from 22 patients were diagnosed to be positive for metastasis adenocarcinoma in CSF. TP with Papanicolau stain showed satisfactory cytomorphology when compared with specimen of CP preparation and cytokeratin immunocytochemical staining. All the TP processed cases belonged to satisfactory/superior categories based on the assessment of technical artifact, which potentially helps in decreasing diagnositc errors. However, in 10 out of 45 cases, diagnostic atypical cells were present only in one of the two slides. Conclusions: Immunocytochemical stain for cytokeratin along with TP processed specimen helps in decreasing potential diagnostic errors in the cytological diagnosis of metastatic carcinoma in CSF specimen.

Fine Needle Aspiration Cytology of Subacute Granulomatous Thyroiditis: A Clinico-Cytological Review of 10 Cases with Immunocytochemical Analysis

Although subacute granulomatous thyroiditis(SGT) is usually diagnosed clinically, papillary carcinoma or other thyroid conditions must be considered in the differential diagnosis. We retrospectively reviewed the clinical and fine-needle aspiration(FNA) cytologic findings seen in 10 SGT cases to decide what are the most reliable cytologic findings and the most helpful molecular tools for reaching a confident cytologic diagnosis. The most representative smear slides were retrieved to perform immunocytochemistry for cytokeratin 19(CK19) and Ret protein. Five papillary carcinomas(PTCs) were included as controls. The constant and typical cytologic findings of SGT were multinucleated giant cells(MGCs) (100%), epithelioid granulomas(90%), an inflammatory dirty background(90%) and plump transformed follicular cells(80%) without fire-flare cells, oncocytic cells or transformed lymphocytes. The immunoreactivities for CK19(37.5%) and Ret(10%) of the follicular cells of SGT were less than those(CK19 and Ret:100%) of PTC. CK19 immunoreactivity of the MGCs was seen in only one case of PTC. There was no significant difference between CK19 and Ret immunocytochemical staining for the MGCs of both SGT and PTC. The results of this study demonstrate that the cytological diagnosis of SGT can be improved by employing a combination of the typical and constant diagnostic cytological features and immunocytochemical results.

Role of fine needle aspiration cytology in the management of pediatric renal tumors

Fine needle aspiration cytology (FNAC) is a useful pre-operative procedure in pediatric renal tumors. It is a safe technique that does not upstage the tumor and permits positive diagnosis of Wilm's tumor in almost all aspirates with use of immunocytochemistry for cytokeratin and WT-1. Advanced information on diagnosis of Wilm's tumor, especially, the presence of stroma predominance or anaplastic change is a guide to selection and monitoring of chemotherapy. Early diagnosis of clear cell sarcoma of the kidney (CCSK) by FNAC helps in the evaluation of bone metastasis and selection of Doxorubicin-based chemotherapy protocols to which the tumor is responsive. Thus, the routine use of FNAC is helpful for further investigation and management of pediatric renal masses.

Utilization of fresh human tympanic membranes for structural analysis and cytokeratin immunocytochemistry implementing resin techniques

Conclusions. The results of this study have demonstrated for the first time that tympanic membrane (TM) structure is preserved following removal of fresh, normal tissue from patients undergoing surgery. Greater clarity has been demonstrated using resin sections than in previous studies on paraffin sections. Of particular note, cytokeratin (CK) immunocytochemistry was successfully performed on resin sections, which has not been previously reported. This may have potential applications for future work involving tissues that express CKs. Objectives. To analyse the structure of normal, fresh human TM specimens after surgical removal and to evaluate their CK immunocytochemistry using resin techniques, neither of which have been demonstrated previously. Material and methods. Seven TM specimens were removed during surgery and then preserved in a modified Karnovsky's fixative. Semi-thin and thin sections were examined by means of light and electron microscopy, respectively. For comparison purposes, paraffin block-embedded specimens were also sectioned. CK immunocytochemistry was performed on semi-thin sections using standard immunoperoxidase techniques, with expression being demonstrated using light microscopy. Results. The three-layer architecture of the TM was preserved. The morphology of the TM was vastly superior in the semi-thin resin sections than in the thicker paraffin sections. The outer, middle and inner layers were clearly demonstrated. The integrity of the outer epithelial layer was maintained, with an outer keratinizing stratum corneum and underlying stratum granulosum, stratum spinosum and stratum basale layers resting on the basal lamina. The thin inner mucosal layer was also viable, consisting of simple squamous or cuboidal cells. Preservation of the middle lamina propria was achieved, with demonstration of the outer radial and inner circular fibres. CK immunocytochemistry utilizing resin techniques provided excellent staining of CK 7 and 8 in the inner layer, with positive staining of CK 5 and 10 in the outer layer.

Reconstruction of epidermis by grafting of keratinocytes cultured on polymer support--clinical study.

Extensive wound coverage still represents a challenge for contemporary medicine. We demonstrate the results of a clinical trial of the grafting of cultured keratinocytes directly on a polymer cultivation support in the treatment of skin defects in seriously burned patients and in patients with trophic ulcers. Wound closure was evaluated clinically. The morphology and phenotypic pattern of the reconstructed epidermis, including the basal lamina, as well as the presence of Langerhans cells, were evaluated immunocytochemically using a panel of monoclonal antibodies. All layers of the reconstructed epidermis were normally differentiated (cytokeratin immunocytochemistry). The basal lamina contained collagen type IV and laminin. The reconstructed epidermis was extensively colonized by Langerhans cells. The results of the described technology are encouraging, especially in patients after a burn injury. The described procedure is suitable for the treatment of skin defects in clinical practice.

The aggressive nature of the odontogenic keratocyst: is it a benign cystic neoplasm? Part 3. Immunocytochemistry of cytokeratin and other epithelial cell markers

Numerous studies of keratin expression by the more common odontogenic cysts were done to determine whether patterns of cytokeratin staining could provide accurate diagnostic markers for the different varieties; to see whether comparative studies with oral mucosa and developing odontogenic epithelium could explain the pathogenesis of the cysts; and whether cytokeratin patterns could provide clues in elucidating the aggressive nature of the OKC. This review was a complex task with a range of at least 19 different cytokeratins being studied and also a broad range of antibodies in use for the same cytokeratin or group of cytokeratins. Moreover, there was not always standardisation of laboratory techniques in the selection and preparation of material. These difficulties were, in general, recognised by the different workers in the field, particularly when there was disagreement on results and caution was expressed about drawing conclusions from some positive findings. It would be fair to conclude that cytokeratin immunocytochemistry has not advanced to any meaningful extent, its use as a diagnostic marker for the OKC nor in eludidating its pathogenesis. With regard to OKC behaviour, it has been pointed out that there was strong reaction of OKC lining for keratin 16, a cytokeratin that has been associated with high proliferative activity. Yet other studies have also shown keratin 16 expression in dentigerous and radicular cysts. Differences in cytokeratin, EMA and CEA immunocytochemical reactivity between the parakeratinised and orthokeratinised varieties of cyst were demonstrated and the suggestion made that the orthokeratinised type has a considerably less aggressive behaviour, is a different entity and should bear a different name. Furthermore, Ki67 positive cells in the parakeratinised OKC linings were considerably more frequent than in the orthokeratinised linings. OKC, dentigerous and radicular cyst epithelium reacted positively for epithelial growth factor receptor (EGFr) but a trend indicating the most intense staining in the OKCs, followed by the dentigerous and then the radicular cyst linings led to the conclusion that the OKCs have an intrinsic growth potential not present in other odontogenic cysts.

Novel adaptation of brush cytology technique for short-term primary culture of squamous and Barrett's esophageal cells

Background: Esophageal disease research is impeded by a paucity of nonmalignant cell lines. Furthermore, culture of primary esophageal cells is difficult because of a lack of cell adhesion and contamination. The aim of this study was to develop a short-term culture method to facilitate cell physiologic studies by using primary esophageal cells. Methods: By using a cytology brush, squamous and Barrett's epithelial cells were obtained from the esophagus of patients undergoing upper endoscopy. Cells were brushed onto chamber slides and allowed to adhere to the surface. Primary culture media was then added and cells were maintained at 37°C for up to 72 hours. Cell yield and viability were calculated after trypan blue staining. For cell physiologic studies, cells were loaded with pH-sensitive dye BCECF-AM and intracellular pH measured on a dual excitation fluorescence microscope after an ammonium chloride prepulse. At the end of the physiologic experiments, cells were fixed in methanol and acetone and the cell type was verified with cytokeratin immunocytochemistry. Results: Viable human esophageal cells were maintained in culture for up to 72 hours. The cells extruded trypan blue, and BCECF-AM was cleaved to BCECF by an intact cell membrane and permitted intracellular pH measurements. The epithelial cell origin of the cells was confirmed by cytokeratin staining. There was no contamination over the culture period and no overgrowth by lymphocytes or fibroblasts. Conclusions: This novel adaptation of brush cytology technique enables short-term culture of esophageal cells for in vitro studies. This rapid, simple primary culture technique is suitable for endoscopy and does not require the immediate, time-consuming laboratory techniques. (Gastrointest Endosc 2001;54:186-9.)

Novel adaptation of brush cytology technique for short-term primary culture of squamous and Barrett's esophageal cells☆, ☆☆, ★

<h2>Abstract</h2> <b>Background:</b> Esophageal disease research is impeded by a paucity of nonmalignant cell lines. Furthermore, culture of primary esophageal cells is difficult because of a lack of cell adhesion and contamination. The aim of this study was to develop a short-term culture method to facilitate cell physiologic studies by using primary esophageal cells. <b>Methods:</b> By using a cytology brush, squamous and Barrett's epithelial cells were obtained from the esophagus of patients undergoing upper endoscopy. Cells were brushed onto chamber slides and allowed to adhere to the surface. Primary culture media was then added and cells were maintained at 37°C for up to 72 hours. Cell yield and viability were calculated after trypan blue staining. For cell physiologic studies, cells were loaded with pH-sensitive dye BCECF-AM and intracellular pH measured on a dual excitation fluorescence microscope after an ammonium chloride prepulse. At the end of the physiologic experiments, cells were fixed in methanol and acetone and the cell type was verified with cytokeratin immunocytochemistry. <b>Results:</b> Viable human esophageal cells were maintained in culture for up to 72 hours. The cells extruded trypan blue, and BCECF-AM was cleaved to BCECF by an intact cell membrane and permitted intracellular pH measurements. The epithelial cell origin of the cells was confirmed by cytokeratin staining. There was no contamination over the culture period and no overgrowth by lymphocytes or fibroblasts. <b>Conclusions:</b> This novel adaptation of brush cytology technique enables short-term culture of esophageal cells for in vitro studies. This rapid, simple primary culture technique is suitable for endoscopy and does not require the immediate, time-consuming laboratory techniques. (Gastrointest Endosc 2001;54:186-9.)

Influence of pigment content, intracellular calcium and cyclic AMP on the ability of human retinal pigment epithelial cells to contract collagen gels

Purpose. The aim of the study was to determine to what extent collagen gel contraction could be reduced by calcium and calmodulin antagonists and agents that elevate cyclic AMP in order to develop a pharmacological approach to prevent/arrest RPE contraction of epiretinal membranes in proliferative vitreoretinopathy. We also explored a possible role of pigment in collagen gel contraction. Method. We measured RPE mediated contraction of 3D collagen gels in the presence and absence of the calcium and calmodulin antagonists TMB8, Verapamil and Tamoxifen and the cAMP elevating agents IBMX and Forskolin. The effect of pigment on collagen gel contraction was assessed by comparing gel contraction mediated by RPE cells re-pigmented with melanin with that mediated by unpigmented RPE. The effect of IBMX on RPE proliferation was assessed using a BrdU ELISA and the effects of IBMX on RPE cytoskeleton and cell shape were assessed using Actin and Cytokeratin immunocytochemistry. Results. We report that both cAMP elevating agents and calcium and calmodulin antagonists reduce RPE mediated collagen gel contraction. Cyclic AMP elevation was more effective than a reduction in calcium in reducing contraction. There were no significant advantages in combining both approaches. The presence of melanin had no effect on gel contraction. Calcium antagonists and particularly agents which elevate cAMP caused RPE cells in collagen gels to extend fewer and shorter processes. cAMP elevation in particular caused RPE cells to become more rounded and develop arborised cell processes. Immunostaining for actin and cytokeratin revealed changes in cytoskeletal organisation in response to IBMX in that cells contained less actin than untreated cells and concentrated cytokeratins more centrally. Conclusion. We have identified two possible pharmacological approaches which may provide a new direction for preventing or slowing down the development of PVR.

Evaluation of Cytolyt and PreservCyt as Preservatives for Immunocytochemistry for Cytokeratin in Fine Needle Aspiration

Evaluation of Cytolyt and PreservCyt as Preservatives for Immunocytochemistry for Cytokeratin in Fine Needle Aspiration

Distribution of cytokeratin markers in Barrett's specialized columnar epithelium

BACKGROUND & AIMS: The cell of origin for Barrett's epithelium is unknown. A multilayered epithelium within Barrett's epithelium has been noted recently. To investigate the hypothesis that this multilayered epithelium may be a transitional stage between squamous and Barrett's epithelium, cytokeratin immunocytochemistry was used to examine normal squamous, Barrett's, and multilayered epithelium.METHODS: Seventeen endoscopic biopsy specimens taken from the squamo-Barrett's junction of 8 patients with Barrett's epithelium and 3 biopsy specimens from the gastroesophageal junction of 3 patients without Barrett's epithelium were investigated. Antibodies to cytokeratins 4 and 13 were used as markers for squamous differentiation, and antibodies to cytokeratins 8 and 19 were used as markers for glandular differentiation. Coded samples were evaluated by immunocytochemistry.RESULTS: In patients with Barrett's epithelium and control patients, staining with columnar markers was confined to either the Barrett's or the gastric columnar epithelium. Staining with squamous markers was confined to the adjacent squamous epithelium. In contrast, focal areas of multilayered epithelium amidst Barrett's epithelium stained with cytokeratin antibodies for both squamous and columnar epithelium.CONCLUSIONS: A focal multilayered epithelium within Barrett's epithelium that expresses concurrently both squamous and glandular cytokeratin markers is described. These findings suggest a multipotential cell as the cell of origin of Barrett's epithelium.(Gastroenterology 1997 Mar;112(3):760-5)

Concurrent fluorescence in situ hybridization and immunocytochemistry for the detection of chromosome aberrations in exfoliated bronchial epithelial cells.

A procedure was developed to allow concurrent detection of chromosome aberrations and identification of bronchial epithelial cells. Fluorescence in situ hybridization for chromosome 7 and immunocytochemistry for cytokeratin were performed on exfoliated bronchial epithelial cells in a sputum sample from a cancer patient. The Spectrum Orange-labeled alpha satellite probe for chromosome 7 produced red fluorescence, nuclei were counterstained with 4,6-diamidino-2-phenylindole (blue), and cytokeratin was visualized using a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (green). This procedure allowed the rapid identification of airway epithelial cells with numerical chromosome aberrations in this sample. Ultimately, this procedure could increase the sensitivity and specificity of sputum cytology as a laboratory diagnostic tool for the early detection of lung cancer.

Cytokeratin distribution and functional properties of growth hormone-producing pituitary adenomas.

In addition to its structural function, cytokeratin may have other important roles within cells. We have reported that in growth hormone-producing adenomas (GH cell adenomas), two distinct types can be recognized by their cytokeratin distribution patterns (dot-like or perinuclear pattern) and that each type has different clinicopathological and endocrinological properties. To confirm these phenomena in a larger series and to clarify the significance of different cytokeratin distribution patterns, we studied cytokeratin localization in 70 GH cell adenomas from acromegalic patients. Type I adenomas ( 15) almost exclusively (>98%) composed of cells with a prominent, dot-like distribution; type 2 adenomas (36) comprised of cells with perinuclear cytokeratin; and type 3 adenomas (11) comprised of both cell types were separated. The remaining 8 did not exhibit a distinct distribution pattern. By electron microscopic immunocytochemistry for cytokeratin, dot-like distribution corresponded to fibrous bodies, whereas perinuclear distribution represented immune deposition in the perinuclear zone. Immunohistochemistry for GH, prolactin, β-thyrotropin, and α-subunit of glycoprotein hormones revealed a reduced expression of these hormones in type 1 adenomas, compared with types 2 and 3 adenomas. In normal pituitary glands, almost all GH cells showed a perinuclear cytokeratin distribution, and only a few GH cells exhibited a dot-like pattern. These findings suggest that a dot-like cytokeratin distribution in GH cells may be pathological (a change from physiological perinuclear distribution) and that adenomas with such a distribution may reduce endocrine activities as a result of unknown factors.