A new technique for hard tissues

Fossil tissues generally require specialized processing. Most thin sectioning techniques yield unstained sections or require unwieldy methods to produce stained sections. I outline here two simple techniques for producing stained, ground, thin sections using readily available Romanowsky-type cytology stains and a urine sediment stain. Results are comparable to hematoxylin and eosin stained specimens.     

Sensitivity and specificity of cytodiagnosis of body fluids in a laboratory of urgencies

Abstract

The main purpose for studying cytological body fluids is confirmation of a benign or malignant effusion. Our cytology laboratory analyzes body fluids and results are requested urgently. The samples are stained by the Giemsa and Papanicolaou methods to give a preliminary report, then they are examined by other complementary techniques. Three hundred thirty samples of pleural and peritoneal fluids were studied to compare the sensitivity of Papanicolaou and Giemsa stains. AgNOR assay, immunocytochemistry and assessment of ploidy were used to improve the sensitivity of the cytodiagnosis. Two hundred one samples were positive, 84 negative and 45 inconclusive using the Papanicolaou stain, while 135 samples were positive, 72 negative and 123 inconclusive using Giemsa stain. The sensitivity was 79%, 53% and 83% for Papanicolaou, Giemsa, and both techniques together, respectively. Using complementary techniques, the sensitivity reached 95% for AgNOR, 87% for tumor markers (panel), and 92% for Ploidy. There were no false positive in our series; therefore specificity was 100%. The use of both Papanicolaou and Giemsa in conjunction increased the sensitivity of the cytodiagnosis in body fluids. The complementary methods, especially AgNOR assay and assessment of ploidy, diminished the number of inconclusive cases.     

The analysis of some commercial dyes and Romamowsky stains by high-performance liquid chromatography.

High-performance liquid chromatography has been used to quantitate batch variations in commericial samples of thiazine dyes, thiazine eosinates, and Romanowsky-type blood stains. It has been observed that all the dyes and eosinates examined, only methylene blue chloride and thionin were reasonably free of their methylated, demethylated, or oxidized homologs. Large variations in composition were observed between most of the samples of each type examined. In several instances the labeled compound was a minority species. In one instance a dye was apparently mislabeled. Large compositional variation was found between various batches of Wright and Giemsa stains, whereas significant differences between the thiazine composition of these two stain types were minor. Very little compositional variation was observed between the lots of LARC stain examined. The thiazine composition of Ames stain was similar for the three lots examined. Ames stain, however, was found to contain several components of unknown composition which have been linked to degradation products formed when stains are aged in methanolic solution.     

Fine needle aspiration cytology of suspected tuberculous lymphadenitis

Fine needle aspiration cytology of suspected tuberculous lymphadenitis The aims of this cross-sectional study were to describe the distributional patterns of tuberculous lymphadenitis and to assess the correlation between fine needle aspiration cytology (FNAC) and the Ziehl Neelsen staining technique in diagnosing tuberculous lymphadenitis. Romanowsky's method (Wright's stain) for cytological diagnosis and Ziehl Neelsen (hot method) for the identification of acid-fast bacilli were utilized. Out of one hundred and twenty-eight consecutive patients attending the cytological diagnostic service of the Department of Pathology within Jimma University, 89 (69.6%) of the patients were younger than 30 years of age. The male to female ratio was 1.3 : 1. The cervical region was the most common site and involved 95 cases (74.2%), followed by the axillary and inguinal lymph node regions (20.3% and 4.3%, respectively). The Wright's-stained cytology smears were grouped into three categories: epithelioid granulomas without necrosis, epithelioid granulomas with caseous necrosis and necrosis without epithelioid granulomas. The Ziehl Neelsen stains were undertaken on separate slides: 20.0% of the cases showing epithelioid granulomas without necrosis, 61.9% of those with epithelioid granulomas with necrosis/abscesses and 69.7% of those with necrosis without granulomas were found to be positive for acid-fast bacilli. The overall positivity for the ZiehlNeelsen stained cases was 59.4%. It can therefore be concluded that FNAC is a reliable diagnostic tool in helping to avert the more invasive surgical procedures undertaken in the diagnosis of tuberculous adenitis. The ZiehlNeelsen stain for identification of acid-fast bacilli should be incorporated as an adjunct to increase the diagnostic accuracy of tuberculous lymphadenitis.     

Adaptations of Goldner's Masson Trichrome Stain for the Study of Undecalcified Plastic Embedded Bone

Specialized adaptations for application of Goldner's Masson trichrome stain to plastic embedded undecalcified bone specimens are presented. This stain can be used successfully on methyl-glycol methacrylate, glycol methacrylate and Spurr embedded bones. The stain affords the advantage of good cellular staining due to the hematoxylin component with concomitant sharp discrimination of mature bone matrix which stains green, immature new bone matrix which stains red, and calcified cartilage which stains very pale green. Use of red filters during photomicrography aids in bone-osteoid discrimination in black and white photographs.     

Demonstration of Urinary Eosinophils in Schistosoma haematobium: a Comparative Study among Three Different Stains

Three stains, Hansel's stain, alkaline erythrocin B (AEB) and naphthalene black (NB), were used to demonstrate eosinophils in the urine of patients infected with Schistosoma haematobium. Hansel's stain was superior to the other two stains; it stained eosinophils bright red and their nuclei faint blue, and they were easily differentiated from neutrophils, lymphocytes, macrophages and epithelial cells. The method using AEB took longer than Hansel's stain and 10% of the specimens were lost during staining with this method. Like eosinophils, the neutrophils took up NB stain and their nuclei stained poorly with the counterstain.     

The Degradation of Romanowsky-Type Blood Stains in Methanol

The oxidative demethylation of Romanowsky-type stains in methanol has been examined quantitatively with respect to its effect upon the staining of blood smears. Spectral changes in hound dye, observed through two color filters, have been measured for the nuclei and cytoplasm of segmented neutrophils and monocytes utilizing the LARC™ automated differential analyzer. Stain decomposition in methanol results in a large loss in staining intensity with little change in color. The loss in intensity has been correlated with the observed spectral changes in the degraded stain. High-performance liquid chromatographic analysis of degraded stain samples has shown the products of methanolic degradation to be different from those obtained in aqueous polychroming reactions. To maintain a stain of defined thiazine dye composition and thus defined staining properties, refrigeration is recommended.     

A New Stain for Pollen Fertility Studies

The juice from the berries of Cocculus hirsutum was extracted and used for pollen fertility studies in various crops. Two stains were prepared: P. H. Ramanjini (PHR) stain and modified PHR stain. The modified PHR stain contains lactic acid and produces the best staining differentiation. The intensity of the staining was dependent on the thickness of the pollen cell walls, hence PHR stain is recommended for thick walled pollen grains and the modified PHR stain for pollen with relatively thin walls. The preparation of both the stains are very simple, quick and inexpensive.     

Impact factor in cytopathology journals: what does it reflect and how much does it matter?

B. AbdullGaffar
Impact factor in cytopathology journals: what does it reflect and how much does it matter? Objective: To study the trends of impact factor (IF) in four cytopathology journals. To investigate the factors that might influence IF in cytopathology literature and whether IF has any impact on cytopathology practice. Methods: The IFs of four cytopathology journals were searched from 2005 to 2009. The IFs and their relationships with the types and number of publications, publishers, the official societies, readership, the quality of their contents, the topics covered and the levels of evidence were compared. Results: Cancer Cytopathology (CC) had the highest IF. Acta Cytologica (AC) had the lowest IF, which appeared to be in decline. Cytopathology (C) and Diagnostic Cytopathology (DC) had a slow but steady increase in their IF. Components that might influence these differences could include the category and the society of the journal, targeted readers and certain types of publications. Publishers, the number of publications, the types of topics covered and the levels of evidence probably have no major effect on IF. Conclusions: IF has its own benefits and original applications. IF is a quantitative measure that does not reflect the levels of evidence in cytopathology journals. IF should not be abandoned because it might encourage competition between cytopathology journals, but it should not dictate their contents.     

Aspergillus in the Papanicolaou stain: morphology, fluorescence and diagnostic feasibility

hettlich c., küpper th., wehle k. and pfitzer p. (1998) Cytopathology 9, 381–388
Aspergillus in the Papanicolaou stain: morphology, fluorescence and diagnostic feasibility
Aspergillus species exhibit a distinct and clear fluorescence in Papanicolaou-stained cytological samples. The Papanicolaou (PAP) stain enhances the autofluorescence of cultured aspergilli and allows better cytological recognition of the fungus by fluorescence microscopy when it is not easily discerned from its surroundings by light microscopy. Morphological properties can be better distinguished and facilitate the differentiation of aspergillus organisms from other filamentous fungi. Neither light nor fluorescence microscopy, the cytological quality nor the presence of phagocytosed hyphae in alveolar macrophages allow distinction between infection and contamination with Aspergillus species. Only the presence of eosinophilic inflammation permits a tentative diagnosis of an Aspergillus infection. In conclusion, PAP fluorescence reduces the need for special stains, is superior to and quicker than other investigative techniques and enhances the sensitivity and specificity of cytological investigation when a rapid and reliable identification of Aspergillus is needed.     

The degradation of Romanowsky-type blood stains in methanol.

The oxidative demethylation of Romanowsky-type stains in methanol has been examined quantitatively with respect to its effect upon the staining of blood smears. Spectral changes in bound dye, observed through two color filters, have been measured for the nuclei and cytoplasm of segmented neutrophils and monocytes utilizing the LARC automated differential analyzer. Stain decomposition in methanol results in a large loss in staining intensity with little change in color. The loss in intensity has been correlated with the observed spectral changes in the degraded stain. High-performance liquid chromatographic analysis of degraded stain samples has shown the products of methanolic degradation to be different from those obtained in aqueous polychroming reactions. To maintain a stain of defined thiazine dye composition and thus defined staining properties, refrigeration is recommended.     

教学用细菌染色方法的改良

常用细菌染色有革兰氏染色,抗酸染色及检测细菌特殊结构的特殊染色法,本文对改良了的染色法进行比较和进一步改进,实验结果证明有操作简单,染色效果好等优点。     

Demonstration of Urinary Eosinophils in Schistosoma haematobium: a Comparative Study among Three Different Stains

Three stains, Hansel's stain, alkaline erythrocin B (AEB) and naphthalene black (NB), were used to demonstrate eosinophils in the urine of patients infected with Schistosoma haematobium. Hansel's stain was superior to the other two stains; it stained eosinophils bright red and their nuclei faint blue, and they were easily differentiated from neutrophils, lymphocytes, macrophages and epithelial cells. The method using AEB took longer than Hansel's stain and 10% of the specimens were lost during staining with this method. Like eosinophils, the neutrophils took up NB stain and their nuclei stained poorly with the counterstain.     

Factors influencing extract of Hibiscus sabdariffa staining of rat testes

Abstract

Some plant extracts can be used in biology and medicine to reveal or identify cellular components and tissues. We investigated the effects of time and concentration on staining of histological sections of rat testes by an acidified extract of Hibiscus sabdariffa. An ethanolic extract of H. sabdariffa was diluted using 1% acetic acid in 70% ethanol to stain histological sections of testes at concentrations of 0.2, 0.1 and 0.05 g/ml for 5, 10, 15, 30, 45 and 60 min. The sections of testes were stained deep red. The staining efficiency of H. sabdariffa was greater at a high concentration and required less time to achieve optimal staining. H. sabdariffa is a strongly basic dye that can be used for various diagnostic purposes. Staining time and concentration must be considered to achieve optimal results.     

The UEMS Section/Board of Pathology,Chapter 6: Requirement for Recognition of Postgraduate Training in Pathology: a presentation of the Paris Document

M. Tötsch, C. Cuvelier, L. Vass and A. Fassina and on behalf of the participants of the UEMS Section/Board of Pathology meeting in Paris 2012
The UEMS Section/Board of Pathology, Chapter 6: Requirement for Recognition of Postgraduate Training in Pathology: a presentation of the Paris Document After more than five years discussion the UEMS Section/Board of Pathology agreed a specification of requirements for recognition of post‐graduate training in pathology, which is the key to the future of our discipline. The document published here, subject to ratification by UEMS Council, was voted on and accepted by the Pathology Board at the UEMS Paris meeting of 9 June 2012. Cytopathology is regarded as integral part of pathology: in general, training in pathology takes five years and maintains a common trunk of four (minimum three) years where surgical pathology, autopsy pathology and basic knowledge of neuropathology, dermatopathology and cytopathology are adequately trained and assessed. Training in so‐called ‘areas of interests’ covers the remaining 12–24 months. Certificates of ‘advanced level of competence’ remain within the authority of national boards. As senior members of its Executive Board, we believe that the European Federation of Cytology Societies (EFCS) should take responsibility for establishing 1) standards in the quality of cytopathology training, 2) training guidelines and qualification for advanced levels of competence in cytopathology, 3) manpower planning, 4) tutorials for pathologists and cytotechnologists and 4) standards of cytotechnologist training.     

The use of new probes and stains for improved assessment of cell viability and extracellular polymeric substances in <Emphasis Type="Italic">Candida albicans</Emphasis> biofilms

Phenotypic and genotypic cell differentiation is considered an important feature that confers enhanced antifungal resistance in candidal biofilms. Particular emphasis has been placed in this context on the viability of biofilm subpopulations, and their heterogeneity with regard to the production of extracellular polymeric substances (EPS). We therefore assessed the utility of two different labeled lectins Erythrina cristagalli (ECA) and Canavalia ensiformis (ConA), for EPS visualization. To evaluate the viability of candidal biofilms, we further studied combination stains, SYTO9 and propidium iodide (PI). The latter combination has been successfully used to assess bacterial, but not fungal, viability although PI alone has been previously used to stain nuclei in fungal cells. Candida albicans biofilms were developed in a rotating disc biofilm reactor and observed in situ using confocal scanning laser microscopy (CSLM). Our data indicate that SYTO9 and PI are reliable vital stains that may be used to investigate C. albicans biofilms. When used together with ConA, the lectin ECA optimized EPS visualization and revealed differential production of this material in mature candidal biofilms. The foregoing probes and stains and the methodology described should help better characterize C. albicans biofilms in terms of cell their viability, and EPS production.     

Fine needle aspiration cytology: a survey of current European practice

Fine needle aspiration cytology (FNAC) is practised widely throughout Europe. The majority of countries have dedicated cytopathologists as well as histopathologists practicing cytology. Despite this, FNAC is performed mostly by clinicians and radiologists except in the larger centres with dedicated staff with a special interest in cytopathology. The advent of One-Stop diagnostic services and image-guided procedures are prompting further development of FNAC clinics where cytopathologists take their own samples, issue reports in the same clinical session and take extra material for ancillary tests to complete the diagnosis. The volume of FNAC work varies accordingly; in dedicated centres FNAC represents up to 80% of the workload whilst, in the majority of countries, it represents one quarter or less. Hence, the rate of inadequate FNAC varies widely, depending on the local sampling policies and the organ, but does not exceed 25% in any of the countries. The most sampled organs are breast and thyroid, followed by lymph nodes. Most countries have dedicated training in cytopathology for pathology trainees, the duration varying between 6 months and 2 years of the total training time. This discussion, focusing on European practices, highlights the heterogeneity of FNAC activity but also its success in many centres where it is practiced to a high standard, particularly in breast, thyroid and lymph node pathology. The relatively high rate of inadequate material in some centres reflects local policies and calls for greater uniformity of FNAC practice, particularly specimen sampling. To achieve this, the future direction should concentrate on specialist training, to include performing as well as interpreting FNAC, as part of the curriculum. Current emphasis on web-based training may not provide first hand experience of the FNAC procedure and should be supplemented by attending FNAC clinics and developing the technique to its full potential.     

Evaluation of stain removal and inhibition properties of eight denture cleansers: an in vitro study

Gerodontology 2012; doi: 10.1111/j.1741‐2358.2011.00522.x
Evaluation of stain removal and inhibition properties of eight denture cleansers: an in vitro study Objectives: To determine the ability of eight denture cleansers to remove and inhibit tea‐stain build‐up on acrylic resin. Materials and methods: In the stain removal study, Perspex^® (cast heat polymerised resin) specimens previously soaked in saliva were stained using multiple exposures of chlorhexidine and tea solutions. Specimens were exposed for 1 min to one of the eight denture cleansers for five cycles, washed and dried and their optical density read on a uv/vis spectrophotometer at 295 nm. In the stain inhibition study, clear specimens were exposed to saliva followed by cleansers then tea solution, for five cycles. The build‐up of stain at each cycle was measured, and differences in optical densities from baseline were calculated. Results: All denture cleansers were significantly more effective than water in removing stain (p < 0.05). There were significant differences in cleaning ability between cleansers (p < 0.001), Dentural^® and Kleenite^® were particularly effective. The stain inhibition experiment showed that most cleansers were significantly more effective than water in inhibiting stain (p < 0.05). There were significant differences in inhibition ability between cleansers (p < 0.01). Kleenite^® and Equate were particularly effective. Conclusions: All denture cleansers had a capacity to remove stain and most had an inhibitory effect on staining. Kleenite^® was particularly effective in controlling stain formation.     

Short Nissl Staining for Incubated Cryostat Sections of the Brain

Nissl stain often binds poorly to cryostat sections which have been incubated in solutions of radiolabeled ligands. Such incubation is used in receptor autoradiography of the brain when using the in vitro method. We have developed a rapid (16 min) modification of Nissl staining for sections that bind stain poroly, e.g., incubated sections. The method stains well sections which cannot be stained with other rapid Nissl staining methods.     

DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.