Improved identification of malignant cells in serous effusions using a small, robust panel of antibodies on paraffin-embedded cell suspensions

OBJECTIVE: To evaluate the expression of these markers individually and to find out which markers would be the most effective in a diagnostic panel to reliably discriminate these lesions. STUDY DESIGN: Sections from cell blocks of these fluids were stained with antibodies against calretinin, EMA, HMFG-2, BerEp4, B72.3 and CEA. A preliminary diagnosis was formulated based on cytomorphologic criteria. Subsequently, results of all 6 immunocytochemical stainings were evaluated, the most effective diagnostic pane of antibodies was proposed and staining results using this panel were compared to results obtained by solely cytomorphologic evaluation and to the ultimate diagnosis. RESULTS: Additional immunocytochemical staining with the proposed panel of calretinin, EMA, HMFG-2 and CEA improved sensitivity for malignancy in general from 78% to 96% and specificity from 73% to 91%. Sensitivity for malignant mesothelioma increased from 45% to 91%, with an increase in specificity from 87% to 96%. Sensitivity for adenocarcinoma decreased slightly from 100% to 92%, but specificity increased from 86% to 100%. Overall, diagnostic accuracy increased from 76% to 94%. CONCLUSION: Immunocytochemical staining of standardized cell block reparations of serous fluid cells with a small panel of 4 antibodies significantly improves diagnostic results compared to cytomorphologic evaluation alone.     

Cytologic study of the tissue repair cells of the uterine cervix. With special reference to their origin.

In order to characterize tissue repair cells (TR) of the uterine cervix and clarify their origin, exfoliated cells obtained after laser conization for early cervical lesions were examined. One specimen was first examined by Papanicolaou staining and then examined for epithelial membrane antigen (EMA) and vimentin by immunocytochemical staining, using a restaining method. The other specimen was observed by phosphotungstic acid-hematoxylin (PTAH) staining. Morphologic findings on TR were investigated together with the histologic findings on the wound healing process. TR were classified morphologically into three groups: stromal (STR), epithelial (ETR) and of unknown origin (UTR). Validity of this classification was confirmed by the findings of immunocytochemical staining with EMA and vimentin. These cells appeared one to eight weeks after laser conization. TR with relatively large nuclei, or atypical TR (ATR), appeared when each type of TR was most plentiful, at two to five weeks. Regarding the origins of each TR, cytologic and histologic findings could be considered to offer evidence that ETR originated with hyperplasia of immature cells of the squamous epithelium or reserve cells below the columnar epithelium. The presence of myofibrils in cytoplasm, demonstrated by PTAH staining for STR and some UTR, strongly suggested the possibility that these cells were myofibroblasts in granulation tissue.     

Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.     

External quality control for immunocytochemistry on cytology samples: a review of UK NEQAS ICC (cytology module) results

I. S. Kirbis, P. Maxwell, M. S. Fle?ar, K. Miller and M. Ibrahim External quality control for immunocytochemistry on cytology samples: a review of UK NEQAS ICC (cytology module) results Objective: To date, external quality control for immunocytochemistry on cytology samples is provided only by the United Kingdom national external quality assessment service for immunocytochemistry and in situ hybridisation (UK NEQAS ICC & ISH). For the purpose of this study a retrospective analysis of a comprehensive collection of quality‐related data regarding immunocytochemistry on cytology samples collected through this service was analysed. Methods: The quality of immunocytochemical reactions, using on‐line collected data, was analysed for the last 23 UK NEQAS ICC cytology module external quality assessments carried out on cytology samples completed in the period from 2004 to 2010. Results: Our study showed that the majority of participants in the cytology module (66%) sent formalin‐fixed paraffin‐embedded (FFPE) tissue sections for assessment as in‐house control slides and only 34% sent cytology slides of various types. The highest UK NEQAS ICC score for the quality of immunocytochemical staining among in‐house control slides was achieved on cell block sections, followed by cytospins, FFPE tissue sections, liquid‐based cytology slides and smears. With regard to fixation, acetone‐fixed slides achieved significantly lower scores than other reported fixatives. The strength of agreement in perception of immunocytochemical staining quality was good between in‐house assessors (Kappa = 0.64) but only fair between in‐house and UK NEQAS ICC assessors (Kappa = 0.22). Conclusions: Good quality of immunocytochemical staining can be achieved on cytology slides prepared and fixed in different ways as well as on cell blocks. Unified criteria for high‐quality immunocytochemical staining and proper internal and external quality assurance could facilitate further improvement and standardization of immunocytochemistry on cytology samples.     

Antiserum to lucifer yellow: preparation, characterization, and use for immunocytochemical localization of dye-filled retinal neurons

We present a new method for the preparation of antisera to Lucifer Yellow, and these antisera are here shown to be particularly suitable for immunocytochemical localization of multiple dye-injected cells in large pieces of vertebrate retina. The method involves the preparation of covalent conjugates of the VS isomer of Lucifer Yellow with keyhole limpet hemocyanin (KLH) or rabbit serum albumin (RSA), and their use as immunogens in rabbits. Both carrier protein conjugates yielded robust antibody responses. Antiserum to the KLH-LY conjugate contained precipitating antibodies against LY and KLH, although activity to the latter did not interfere with immunocytochemical staining. Rabbit antiserum to the RSA-LY conjugate contained precipitating antibody only against LY. When used for immunocytochemical staining of large retinal pieces containing many LY-filled cells, both antisera yielded well-stained, darkly filled cells similar to those seen with the Golgi technique; even very fine dendritic processes of retinal ganglion cells could be followed for long distances. LY immunocytochemistry provides a useful alternative to photooxidation for the analysis of multiple dye injected cells, especially in whole mounts. This approach may also be useful for immunocytochemical identification of cells filled with LY after tissue fixation.     

Usefulness of estrogen receptor detection using archival Papanicolaou-stained smears.

OBJECTIVE: To examine estrogen receptor (ER) detection using cytologic specimens and to compare the results with those obtained by the dextran-coated charcoal (DCC) method and enzyme immunoassay (EIA). STUDY DESIGN: Immunocytochemical staining was conducted on 60 cases of breast cancer resected at our hospital between April 1993 and November 1997 in which ER had been measured by DCC or EIA. Specimens for immunocytochemical staining were prepared by a cell transfer method using archival Papanicolaou-stained imprint smears, and ER staining was performed by the labeled streptavidin method using an anti-ER monoclonal antibody. These results were compared with those obtained by DCC or EIA. RESULTS: In immunocytochemical staining for ER, positive staining was observed in the nuclei of tumor cells. A good correlation was obtained between the immunocytochemical staining results and biochemical results. Five cases were positive in anti-ER staining but negative in biochemical tests, and two cases were negative in anti-ER staining and positive in biochemical tests. CONCLUSION: Unlike biochemical assays, the immunocytochemical method does not necessitate use of fresh frozen materials and can be performed even using archival Papanicolaou-stained smears. Immunocytochemical study is a highly useful method for routine ER determination.     

Thyroid Hormone-Induced Maturation of Astrocytes Is Associated with the Expression of New Variants of Vimentin and Their Phosphorylation

The role of thyroid hormone (TH)-induced vimentin expression in promoting the maturation of astrocytes has been investigated. Comparative immunocytochemical staining with vimentin antibody demonstrated distinctly different patterns of expression of vimentin during the progressive maturation of astrocytes of normal and TH-deficient astrocyte cultures. [35S]Methionine labeling of cells showed a significant decline in the level of vimentin in the hypothyroid cultures at all ages from day 5 to 20. Western blot analysis suggested that the maturation of normal astrocytes was associated with the appearance of several acidic and basic variants of vimentin, whose expression was significantly delayed or reduced in the TH-deficient astrocytes. In normal astrocyte cultures, two acidic variants of vimentin, one of similar molecular weight and the other of lower molecular weight, were found to be phosphorylated during the final transformation of the epithelioid form into the mature stellate form. None of these phosphorylated forms was evident in the TH-deficient astrocytes. This was further confirmed by negative immunocytochemical staining of 5- to 18-day-old cultures of TH-deficient astrocytes with antibody TM71, specific to phosphorylated vimentin, compared with age-matched normal astrocytes, which displayed consistent positive staining. The overall results indicate that TH-induced expression of phosphorylated forms of vimentin may be essential for the intracellular organization of the cytoskeleton in a way conducive to the morphological maturation of astrocytes.     

Immunocytochemical detection of estrogen receptors by staining with monoclonal antibodies on cytologic specimens of human breast cancer

A study was undertaken to test the possibility of determining the estrogen receptor (ER) content in human breast cancers by staining with commercial specific monoclonal antibodies (MAbs) on cytologic specimens (touch imprints and fine needle aspirates). The aspirates were suspended in a cell culture medium and cytocentrifuged onto slides to preserve their morphologic characteristics and to allow a proper immunocytochemical staining for ERs. MAb staining for ER was also performed on the respective surgical samples. The staining of cytologic samples for ER showed 100% specificity and 95% sensitivity in comparison to the staining of the histologic samples. Moreover, comparison of the percentage of stained cells in the cytologic specimens to the ER content in the respective surgical specimens, as assayed by the dextran-coated charcoal method, showed the MAb staining of cytologic samples to have 94% specificity and 100% sensitivity. These results support the reliability of MAb staining for ERs in cytologic samples and suggest that it could be the assay of choice in particular clinical settings in the evaluation of primary and recurrent breast cancers.     

Human breast epithelial xenografts: An immunocytochemical and ultrastructural study of differentiation and lactogenic response

Fragments of ductal and lobular epithelium ('organoids') produced by collagenase digestion of reduction-mammoplasty specimens were injected into athymic 'nude' mice. These heterospecific tissues were accepted without rejection, and the presence of xenografts was confirmed by cytology, immunocytochemistry and chromatin staining. Lactation, as confirmed by immunocytochemical and ultrastructural criteria, was observed in the grafted human epithelium during murine pregnancy at both intra- and extra-mammary sites.     

Immunocytochemical staining of effusions; an external quality control study in The Netherlands

Immunocytochemical staining of effusions; an external quality control study in The Netherlands
In The Netherlands an external quality control study of immunocytochemical (IC) staining of effusions was initiated, consisting of three test rounds. The 12 participating laboratories received samples of malignant effusions (runs 1, 2 and 3), and five unstained control specimens prepared from the same material in runs 2 and 3. The laboratories used their own protocols to prepare and stain the samples ('in‐house' specimens). Two persons viewed and scored the slides following preset criteria concerning number and morphology of diagnostic cells, background staining and staining specificity. Better scoring results were found for control specimens, compared with 'in‐house' specimens, primarily caused by cell loss in the latter. This finding underlines the view that high quality IC needs well organized processing and staining procedures, and warrants external quality control systems.     

Aspiration cytology, immunocytochemistry and electron microscopy of a malignant peripheral neuroectodermal tumor. A case report

The findings of fine needle aspiration (FNA) cytology, immunocytochemical staining and electron microscopy (EM) in a case of malignant peripheral neuroectodermal tumor (PNET) presenting as a soft tissue mass in the lateral abdominal wall are reported. The immediate evaluation of the aspirate revealed cells of a small round cell malignant tumor. To provide a specific preoperative diagnosis, additional cytologic material was aspirated for immunocytochemical and ultrastructural investigations. While the results of EM were nonspecific, allowing only the exclusion of other small round cell malignancies, immunocytochemical staining of the aspirate was suggestive of a PNET. The diagnosis of PNET was corroborated by histopathologic and immunohistochemical findings. This case indicates that an exact preoperative categorization of small round cell malignant tumors can be made by FNA biopsy in otherwise equivocal cases when immunocytochemical and ultrastructural techniques are also utilized.     

Atypical Mesothelial Cells Associated With Eosinophilic Pleural Effusions: Nuclear Dna Content and Immunocytochemical Staining Reaction With Epithelial Markers

The nuclear DNA contents of atypical mesothelial cells from five patients who had an eosinophilic pleural effusion (EPE) were studied by the use of DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) DNA staining. Analysis of the nuclear DNA content revealed a polyploid pattern, with a major peak in the tetraploid region. Using an immunocytochemical technique, the atypical mesothelial cells showed a positive reaction for cytokeratin. In contrast carcinoembryonic antigen (CEA) was always negative in these cells. It is suggested that the atypical mesothelial cells with EPE had a higher rate of proliferation than did the normal mesothelial cells.     

Bodian's silver impregnation of endocrine cells

Summary We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

Bodian's silver impregnation of endocrine cells. A tentative explanation to the staining mechanism

We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

Immunocytochemical staining of ovarian cyst aspirates with monoclonal antibody against inhibin

mccluggage w. g., patterson a., white j. and anderson n. h. (1998) Cytopathology 9, 336–342
Immunocytochemical staining of ovarian cyst aspirates with monoclonal antibody against inhibin
Inhibin is a peptide hormone which is produced by ovarian granulosa cells during normal follicular development. It is important that granulosa cells are recognized in fine needle aspirates (FNAs) of ovarian cystic lesions, as this allows definite recognition of a functional cyst and exclusion of a potentially neoplastic epithelial lined cyst. Occasionally the distinction between granulosa and epithelial cells may be difficult, especially when aspirates from functional cysts are unusually cellular. In the present study, FNAs from 33 ovarian cystic lesions were immunostained with a monoclonal antibody against inhibin. Nine cases of peritoneal fluid containing malignant cells in patients subsequently confirmed to have ovarian adenocarcinoma were also stained. Where possible the cytological and immunocytochemical findings were correlated with subsequent biopsy. In most cases in which cytology suggested a functional cyst there was a strong positive staining with anti-inhibin, although occasional cases were negative. One case originally thought to contain epithelial cells stained strongly positive with anti-inhibin and on review was felt to represent a cellular functional cyst. In all other cases where cells were considered to be epithelial there was no staining with anti-inhibin. The study shows that immunocytochemical staining with anti-inhibin may be of value in confirming the presence of granulosa cells, thus establishing a diagnosis of functional cyst. Although negative staining does not exclude a functional cyst, positive staining with anti-inhibin allows exclusion of an epithelial lined cyst and may avoid unnecessary surgical intervention.     

肌管细化是失重性肌萎缩结构和功能变化的基础

本文通过新生大鼠原代培养获取骨骼肌肌管,利用水平回转器模拟失重效应,通过F-actin/G-actin以及F-actin/pERK免疫细胞化学染色,观察研究了模拟失重对肌管形态、微丝及磷酸化ERK表达的影响。通过建立的航天飞行失重性肌萎缩的细胞学模型研究发现:回转后肌管变细,F-actin染色减弱伴有G-actin染色增强,同时pERK染色减弱。表明回转模拟失重条件下肌管发生萎缩、微丝解聚并伴随信号转导活性分子磷酸化ERK表达下降。提示肌管细化是失重性肌萎缩结构和功能变化的结构基础     

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.     

slit: an EGF-homologous locus of D. melanogaster involved in the development of the embryonic central nervous system

A family of loci homologous to the EGF-like portion of Notch, a gene involved in neurogenesis, have been identified in D. melanogaster. The sequence, spatial, and temporal distribution of both RNA and protein of one of these loci suggest a possible role in the development of the central nervous system (CNS). In situ hybridization and antibody staining of embryos show initial localization in cells along the midline of the neuroepithelium. High level expression is restricted in the developing embryo to a subset of six midline glial cells abutting growing axons. Extracellular localization is suggested by the presence of EGF-like repeats in the deduced protein sequence and antibody staining. Cytological, immunocytochemical, genetic, and molecular data show that this gene corresponds to the slit locus. Mutations in this locus result in the collapse of the regular scaffold of commissural and longitudinal axon tracts in the embryonic central nervous system.     

Immunocytochemistry: its evolution and criteria for its application in the study of epon-embedded cells and tissue

The word immunocytochemistry is currently used to describe a number of methods that can be employed to localize antigens within cells by means of antigen-specific antibodies. In this article we will review a number of these methods, including immunofluorescence, immunoperoxidase, avidin-biotin, and colloidal-gold techniques. The advantages and disadvantages of the various methods are discussed, special attention being focused upon immunocytochemical staining of plastic-embedded tissue. Studies on the light microscope level show that embedding tissue in plastic prior to immunoperoxidase staining not only improves visualization of antigen-specific staining but also provides an accurate and efficient means of prescreening tissue for antigen prior to immunocytochemical staining on the electron microscope level. Varying section thickness between 1 and 3 microns does not significantly influence staining, whereas the fixative used to preserve the tissue under study does. On the electron microscope level, the colloidal gold technique appears superior to immunoperoxidase staining. It is both esthetically more pleasing and highly sensitive. Of five different colloidal gold methods tested, the most sensitive is the two-step technique that employs an antigen-specific primary antibody followed by a gold-labeled secondary antibody. Throughout this article, special emphasis is placed on the use of proper controls, both on the light and electron microscope levels. Where possible, such controls should include substitution of specific antiserum with normal serum; the use of antigen-adsorbed antiserum; the use of antisera with specificities for antigens not present in the tissue being studied; the use of tissue previously shown to be stainable for the antigen; and if cultured cells are being studied, the use of a number of cell types that do not contain the antigen.     

Immunocytochemical and Biochemical Analysis of Carbonic Anhydrase in Primary Astrocyte Cultures from Rat Brain

Carbonic anhydrase (CA) was studied in primary monolayer cultures from neonatal rat cerebral hemispheres with both immunocytochemical and biochemical techniques. In such cultures, which consist predominantly of astrocytes, immunocytochemical staining for CA using antibody raised against the type II enzyme from rat erythrocytes resulted in positive staining of the flat, glial fibrillary acidic protein-positive, astrocytic monolayer. Smaller, process-bearing, round cells that grew on top of the astrocytes stained intensely for CA. We estimated that these cells represented 1% or less of the total cells in the cultures, and they have been identified by others as oligodendrocytes. The intensity of the staining of astrocytes for CA could be increased to that observed in oligodendrocytes when the astrocytes were made to round up and form processes by treatment with 2',3'-dibutyryl cyclic AMP. Enzymatic assays showed that CA activity of the cultures after 3 weeks of growth was 2.5- to 5-fold less than that found for cerebral homogenates from perfused 3-week-old rat brains. However, both activities were totally inhibited by acetazolamide with an I50 of 10(-8) M, confirming that both rat brain and the astrocyte cultures possess the high-activity type II enzyme. CA-II activity was unaffected by treatment of the cultures with a method reported to remove oligodendrocytes. Thus, the immunocytochemical and biochemical studies reported here demonstrate that astroglial cells in primary cultures from neonatal rat brain contain CA-II.