Acriflavine-Feulgen stilbene staining: a procedure for automated cervical cytology with a television based system (LEYTAS).

A sample preparation and staining procedure for automated cytology with a TV based system (LEYTAS) is described. It consists of a centrifugation technique and automated acriflavine-Feulgen stilbene staining of cervical specimens. The advantages of using both the fluorescence and the absorption image of acriflavine-Feulgen stilbene stained cervical cells for a television based system are discussed.     

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.     

Planimetry of bronchoalveolar macrophages. Importance of preparation and staining techniques.

Bronchoalveolar lavage seems a well-established, valuable research tool in the study of alveolar macrophages. The influence of fixation, cytocentrifugation and staining procedures on the cellular and nuclear size has been investigated by planimetry. As a reference, mean profile areas of 109 and 39 microns 2 for cell and nucleus, respectively, were measured for alveolar macrophages suspended in the hemocytometer. For comparison, stained Cytospin preparations were measured. Unfixed cells were compressed during cytocentrifugation. The cellular profile areas for Cytospin preparations increased about 15% and 70% after May-Grünwald-Giemsa and Feulgen staining, respectively. The nuclear area was approximately 25% larger for both staining procedures as compared to the hemocytometer values. When the cells had been fixed prior to cytocentrifugation, these differences were less conspicuous. No significant differences were observed after May-Grünwald-Giemsa staining, showing a cellular area of 114 microns 2 and a nuclear area of 45 microns 2. Depending on the staining procedure, low nucleus:cell ratios (31%) were observed after Feulgen staining, while higher values (about 43%) were measured after May-Grünwald-Giemsa staining, regardless of which fixation or centrifugation procedure had been followed. In conclusion, these findings indicate that fixation should be carried out in order to prevent cell changes resulting from cytocentrifugation. Moreover, different staining procedures considerably influence the measurement of cellular and nuclear profile areas and the determination of nucleus:cell ratios.     

Density distribution and immunological reactivity of tumor cells from peritoneal effusions of patients with ovarian neoplasms.

Ascitic samples from 19 patients with primary ovarian non-mucinous carcinomas, three with Krukenberg tumors and eight with noncancerous peritoneal effusions were studied by conventional cytology and immunocytochemical staining. Density gradient centrifugation was applied to fractionate ascitic fluid cells. The enrichment of cell types by this method facilitated their cytomorphological characterization and identification of neoplastic cell subpopulations existing in peritoneal effusions. Immunophenotypic studies of cells were made using monoclonal antibodies (mAbs) against ovarian carcinoma-associated antigens (OC 125, 10B, 8C) and carcino-embryonic antigen (CEA). Non-specific cross-reacting antigen (NCA) was applied as a marker for granulocytes which often accompany peritoneal effusions. Our results indicated that immunofluorescence (IF) staining contributed to the distinction between the primary and secondary ovarian carcinomas. Density gradient centrifugation appeared to be a useful method for separation of mesothelial cells.     

Hydrogenosomes in the rumen entodiniomorphid ciliate Polyplastron multivesiculatum

The rumen entodiniomorphid ciliate protozoon Polyplastron multivesiculatum was shown, by biochemical and electron microscopic techniques, to possess hydrogenosomes. After differential centrifugation of whole cell homogenates the hydrogenosomal marker enzymes pyruvate:ferredoxin oxidoreductase and hydrogenase were recovered predominantly (61% and 70% of activity respectively) in the large granular fractions that were sedimented by centrifugation for 10(4) g-min (fraction P1) and 10(5) g-min (fraction P2). These subcellular fractions contained membrane-bound organelles that were approximately 0.4-0.6 microns in diameter and which had a mean equilibrium density of 1.22-1.24 g ml-1 after isopycnic centrifugation in sucrose gradients. Malate dehydrogenase (decarboxylating) activity, however, was predominantly non-sedimentable after centrifugation for 6 x 10(6) g-min. Numerous hydrogenosome-like organelles were present in the ectoplasm and endoplasm of the cell. Hydrogenase activity was demonstrated and localized in the protozoan cell using a novel staining procedure with distyryl nitroblue tetrazolium chloride (DSNBT).     

A new method for the demonstration of the Bordetella bronchiseptica capsule in electron microscopy

A new and rapid working method is presented for electronmicroscopic preparation of capsules from gram negative bacteria, e.g. Bordetella bronchiseptica and Pasteurella multocida. The advantage of the new technique is the availability of the results within 30 min after starting the preparation. The staining of the capsule by alcian blue has to be done as a first step together with glutaraldehyde fixation, before staining the bacterial cell with phosphotungstic acid. The new staining technic also reveals structural details of the capsule. The described procedure was found to be useful in controlling the development of the bacterial capsule depending on culture media for propagation and maintenance of the above mentioned bacteria.     

Standardized thionin-eosin stain in bronchial cytology. A substitute for hematoxylin-eosin Y staining

A standardized thionin-eosinic acid stain was developed as a quick and highly reproducible staining method for bronchial cytology. Bronchial smears and paraffin-embedded sputum samples were stained with thionin-eosin and with the conventional hematoxylin-eosin Y. Spectral absorption characteristics and staining intensity of thionin-eosin-stained cells were investigated by means of cytophotometry. The staining pattern of thionin-eosin is very close to that of the hematoxylin-eosin stain; the contrast between nucleus and cytoplasm is significantly higher for thionin-eosin. Thionin-eosin can be used for "dye-fixation" of cytologic smears and tissue imprints. Blueing and differentiation (as for hematoxylin-eosin) is not required for thionin-eosin; thus, fixation and staining can be performed within two minutes. The spectral absorption characteristics of thionin-eosin allow reliable automated cytophotometric discrimination of cell nuclei and cytoplasm. The standardized thionin-eosin stain is recommended as a substitute for the hematoxylin and eosin stain in bronchial cytology.     

Increasing diagnostic accuracy with a cell block preparation from thin-layer endometrial cytology: a feasibility study

OBJECTIVE: To investigate (1) the feasibility of preparing cell blocks by inverted filter sedimentation (IFS-CB) from endometrial samplings processed by the ThinPrep (TP) technique (Cytyc Corp., Boxborough, Massachusetts, U.S.A.), and (2) the possibility of increasing the diagnostic accuracy of TP endometrial cytology by examining the tissue architecture as an adjunctive method of detecting endometrial lesions. STUDY DESIGN: Three hundred one endometrial samplings were obtained, using the Endogyn endometrial device (Biogyn S. n.c., Italy), from perimenopausal and postmenopausal women. The endometrial samplings were collected in a vial with liquid fixative for the TP processing. One TP slide was prepared from each case. If adequate material remained in the vial after the TP slide preparation, it was processed for IFS-CB preparation. RESULTS: IFS-CB preparation was processed in 263 cases (87%) with adequate material. Diagnoses on IFS-CB preparations obtained by endometrial sampling matched those of the hysterectomy specimens. The addition of IFS-CB histology to the cytologic diagnosis by TP increased the diagnostic accuracy of endometrial cytology to 96.3% and 100% for benign/atrophic endometrium and adenocarcinoma, respectively (p = 0.39 and 0.46). In hyperplasia without atypia and hyperplasia with atypia, the diagnostic accuracy increased significantly, to 96% and 95.3%, respectively (p = 0.037 and < 0.001). CONCLUSION: This study illustrates the merit of linking TP cytology with direct endometrial sampling, including small tissue fragments and material adequate for IFS-CB preparation. TP cytology provides an accurate cytologic diagnosis and the possibility of IFS-CB preparation, which could be a valuable diagnostic adjunct to TP cytology.     

Method for simple and rapid enumeration of total epiphytic bacteria in the washing solution of rice plants

The phyllosphere is one of the most common habitats for terrestrial bacteria. However, little is known about the populations of bacteria, including unculturable bacteria, that thrive on plant surfaces. Here, we developed a fluorescent nuclear staining technique to easily and rapidly observe and enumerate populations of total and living epiphytic bacteria, with particular emphasis on the concentration by centrifugation and fixation of the epiphytic bacteria. An investigation on the optimal conditions for centrifugation and fixation revealed that centrifugation at 20 400g for 2 min and fixation with 0.5% glutaraldehyde solution were the optimum conditions for observation of the bacteria. Using this technique, we assessed the populations of the total and living bacteria on the surface of rice plants. When epiphytic bacteria were recovered from rice seeds (Oryza sativa 'Koshihikari'), the number of total and living bacterial cells was 7.36 and 6.85 log??·g?1 (fresh mass) in the seed washing, respectively. In contrast, the numbers of total and living bacterial cells in the leaf sheath washings were 5.5-5.8 and 5.3-5.7 log??·g?1, respectively. Approximately 5%-30% of the total bacteria in the washing solution of rice plant were culturable. The usefulness of the enumeration method and the amount of bacteria on the plant surfaces are discussed.     

A standardized pre-treatment method of biofilm flocs for fluorescence microscopic characterization

The availability of epifluorescence microscopy opens the door for the visualization of biofilm flocs via the fluorescence staining technique. However, one of the main problems in the application of the method is a missing standardized protocol for sample preparation. In this study, different pre-treatment methods for the simultaneous staining of different sludge flocs components e.g. extracellular polymeric substances (EPS) and microorganisms (MO) were compared. A method was chosen, which remains the shape of sludge flocs from a municipal wastewater treatment plant and allows to stain major parts of the flocs thoroughly: For this the surrounding wastewater was removed by centrifugation. Then the flocs were resuspended in sterile tap water, immobilized by drying on a diagnostic slide and fixated with formaldehyde. The staining of the different compounds was performed with tetramethylrhodamine isothiocyanate-labeled lectin Concanavalin A (TRITC-Con A; glycoconjugates), fluoresceinisothiocyanate isomer I (FITC; amino groups) and 4,6-diamidino-2-phenylindoldihydrochloride (DAPI; nucleic acids). This staining method was successfully applied for protein- and carbohydrate-quantification using gelatin mixtures of dextran and bovine serum albumin (BSA). The pre-treatment and staining methods were appropriate to compare the composition of different biofilm flocs such as nitrifying activated sludge from a municipal wastewater treatment plant and a pure culture of the bacterial strain Pseudomonas putida (ATCC 17514) as revealed by the results of conventional analytical methods.     

A standardized pre-treatment method of biofilm flocs for fluorescence microscopic characterization

The availability of epifluorescence microscopy opens the door for the visualization of biofilm flocs via the fluorescence staining technique. However, one of the main problems in the application of the method is a missing standardized protocol for sample preparation. In this study, different pre-treatment methods for the simultaneous staining of different sludge flocs components e.g. extracellular polymeric substances (EPS) and microorganisms (MO) were compared. A method was chosen, which remains the shape of sludge flocs from a municipal wastewater treatment plant and allows to stain major parts of the flocs thoroughly: For this the surrounding wastewater was removed by centrifugation. Then the flocs were resuspended in sterile tap water, immobilized by drying on a diagnostic slide and fixated with formaldehyde. The staining of the different compounds was performed with tetramethylrhodamine isothiocyanate-labeled lectin Concanavalin A (TRITC-Con A; glycoconjugates), fluoresceinisothiocyanate isomer I (FITC; amino groups) and 4,6-diamidino-2-phenylindoldihydrochloride (DAPI; nucleic acids). This staining method was successfully applied for protein- and carbohydrate-quantification using gelatin mixtures of dextran and bovine serum albumin (BSA). The pre-treatment and staining methods were appropriate to compare the composition of different biofilm flocs such as nitrifying activated sludge from a municipal wastewater treatment plant and a pure culture of the bacterial strain Pseudomonas putida (ATCC 17514) as revealed by the results of conventional analytical methods.     

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

Summary In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO₄ post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.     

Cytological diagnosis of basal cell carcinoma and actinic keratosis, using Papanicolaou and May–Grünwald–Giemsa stained cutaneous tissue smear

Objective:  Cytology may become the diagnostic method of choice with the advent of new non-invasive treatments for non-melanoma skin cancer, as the sampling technique for cytology entails little tissue disfiguration. The aim of this study was to compare and evaluate the diagnostic performance of scrape cytology using two different cytological staining techniques, and to evaluate additional touch imprint cytology, with that of histopathology of basal cell carcinoma (BCC) and actinic keratosis (AK).
Methods:  We investigated 50 BCC and 28 AK histologically verified lesions, from 41 and 25 patients, respectively. Two separate skin scrape samples and one touch imprint sample were taken from each lesion. The smears were stained with Papanicolaou (Pap) or May–Grünwald–Giemsa (MGG) stains. All cytological specimens were examined in random order by pathologists without knowledge of the histology. Cytodiagnostic results were compared with the histopathological report.
Results:  Scrape cytodiagnosis agreed with histopathology in 48 (Pap) and 47 (MGG) of the 50 BCC cases, and in 26 of 28 (Pap) and 21 of 26 (MGG) AK cases, yielding sensitivities of 96%, 94%, 93% and 81%, respectively. No significant difference in sensitivity between the two staining methods was found but a trend towards higher Pap sensitivity for AK was noted ( P  =   0.10). Touch imprint cytology confirmed histopathology in 38 of the 77 cases of BCC and AK.
Conclusion:  Cytological diagnosis with either Pap or MGG stain for BCC and AK is reliable, and differentiates well between BCC and AK. Imprint cytology proved to be non-diagnostic in half of the examined cases.     

A new type of two-color fluorescence staining for cytology specimens.

A new two-color fluorescence staining technique for cervical cytology specimens is described. To permit application of this staining in automated cytology, techniques for specimen collection and cell preparation giving a sufficient number of well-separated cells on slides were used. The staining consists of a combination of a modified Feulgen-acriflavine procedure for DNA and a primulin or stilbene isothiocyanate staining for protein. This results in a bright yellow nuclear fluorescence and a blue cytoplasmic fluorescence. The staining procedure can be completed in about 90 min and is therefore suitable for routine application. Sequential inspection of the yellow nuclear and blue cytoplasmic fluorescence can be done with the two-wavelength excitation method used in fluorescence microscopy. For the application of this method, special vertical illuminators are now available. These illuminators are provided with quickly interchangeable filter sets permitting consecutive visualization of, for example, only the nuclei in the first image and the whole cell in the second image. This procedure opens new possibilities for rapid image-analysis systems.     

Cotton block method: one-step method of cell block preparation after fine needle aspiration

OBJECTIVE: To describe the cotton block method, an easy, inexpensive, 1-step method of obtaining a cell block after fine needle aspiration biopsy. STUDY DESIGN: Before connection to a 10-mL syringe, the plastic hub of a 22-23-gauge needle is filled with the 3-4-mm, woolly tip of a cotton bud. Aspiration is performed as described elsewhere. After smear preparation, the material remaining in the needle and the material retained in the cotton wool mesh are immediately fixed by aspiration ofa fixative fluid (70% alcoholic formaldehyde-acetic acid). After fixation, the cotton tip is removed and routinely processed for paraffin embedding, and the sections are stained by routine methods used in cytopathology. RESULTS: The results demonstrate that the amount and quality of material obtained in the cotton wool tip is similar to that in the traditional cell block obtained from the pellet after centrifugation of aspirated fluid. CONCLUSION: The method is easy to perform and cost effective and is a rapid way to prepare cell blocks of high quality, allowing special staining techniques and improving cytohistologic correlation.     

Standardization of the Feulgen reaction: the influence of chromatin condensation on the kinetics of acid hydrolysis.

The purpose of the present study was to investigate the influence of chromatin compactness on the kinetics of acid hydrolysis in the Feulgen reaction in cytology. Tissue imprints of rabbit liver, of human bronchial carcinoma and of human blood smears, fixed with alcohol, formaldehyde or with B?hm's solution with and without prior air drying, were stained with a standardized pararosanilin-Feulgen reagent. The time for hydrolysis varied between 7.5 and 120 min. The integrated optical density (IOD) of the cell nuclei was measured with an image analyzer (IBAS 2000). Cells with condensed chromatin (lymphocytes, small cell carcinoma, formaldehyde fixed cells) showed a slow increase of staining intensity and late plateau phase as compared with cells with decondensed chromatin. DNA in condensed nuclei was less susceptible to acid hydrolysis. The degree of chromatin compactness which determines the sensitivity of DNA to hydrolysis is influenced by the type of fixation, cell type and by the functional status of the cell. The conclusion is that Feulgen staining intensities of cells with different degrees of chromatin compactness cannot be compared unless measured in the respective plateau phases of the relevant hydrolysis curves which must be determined individually for each cell type.     

Evaluation of brushing cytology in the diagnosis of Helicobacter pylori gastritis

OBJECTIVE: To evaluate clinical utility of rapid urease test (RUT), brash cytology and histology for detecting Helicobacter pylori. STUDY DESIGN: Brush cytology materials were obtained from the antrum of the stomach in 109 patients who suffered from dyspepsia and were candidates for endoscopy. RUT and histology with hematoxylin-eosin staining were performed. Infection status was established by observation of typical HP in cytology or biopsy. RESULTS: A total of 78% ofpatients were diagnosed as positivefor HP organisms using brush cytology; 66% had histologic results positive for HP and 59% for RUT. Observation of typical organism by cytology or histology was the gold standard; 3 tests results were compared. Sensitivity of brush cytology (95%) was higher than that of histology (80.5%) and RUT (72%). CONCLUSION: Gastric brushing cytology provides a sensitive, inexpensive, accurate and easy technique for rapid detection of HP infection. When additional information on severity of mucosal damage or presence of cell atypias is not necessary, histologic examination can be omitted; a cost-effective strategy for assessing HP status might consist of taking antral biopsies, the former for RUT, and performing brush cytology slides, which should be stained and examined only when the RUT result is negative.     

Immunocytochemical detection of prognostic markers in breast cancer; technical considerations

The purpose of this study was to establish a good technical procedure for immunocytochemical (IC) staining of prognostic markers in breast cancer specimens. The influence of various preparation, fixation and storage methods on ER, P53 and Ki-67 IC staining was assessed, using cells of two breast cancer cell lines T47D (ER/P53+) and ZR-75-ER (ER+, P53-). In addition we searched for a suitable transport medium. Depending on the technical procedure, great variations in expression of the tested antigens were found. Cytospins fixed and stored according to the Abbott method gave the best results. Histocon appeared to be the medium of choice. A good concordance of IC and immunohistochemical (IH) results was found when the adopted method was tested on material of 10 breast cancers. This study underlines the importance of quality controlled standardization of cell processing, fixation and storage of fine needle aspiration (FNA) aspirates in order to obtain reproducible and consistent IC results.     

Preparation of monolayer smears from paraffin-embedded tissue for image cytometry

A method is described for the preparation of monolayer smears from paraffin-embedded tissue suitable for automated image analysis and DNA measurements. The proposed technique uses enzyme treatment and syringing for cell dispersal. Slide preparation is performed by centrifugal cytology. After Feulgen staining the quality of the monolayer smears is sufficiently high to enable visual morphologic evaluation. Automated DNA measurements using the Leyden television analysis system (LEYTAS) show coefficients of variation (CV) of 4.5% for the diploid cell population of the suspended tissue. This is approximately the same as the CV in fresh material from the same tumor. Formalin fixed trout red blood cells are used as reference cells. By applying image cytometry to paraffin-embedded tissue this method allows retrospective studies of, for instance, the significance of DNA content with regard to the behavior of a tumor.