Protein isolation from ear wax made easy.

Cerumen is a waxy substance with a mixture of different lipids and and not yet identified proteins. Analysing ear wax can be quite laborious because of the different and sometimes interfering components. Therefore, time-consuming techniques such as chromatography or spectrometry were used to gain informations about the components of ear wax. Conclusions were drawn from immunohistochemical detections of special proteins within the skin or the glands of the external ear canal about the existence of these proteins within the ear wax. But directly analysing the proteins within the ear wax was difficult. We, therefore, worked out a method to isolate proteins from ear wax. Ear wax was collected from 16 adults with no infections of the external ear canal. The protein isolation was conducted using the Qproteome Mammalian Protein Prep Kit by Qiagen in two different kind of ways (cell and lysat fraction). Afterwards, we performed a quantification of the total protein concentration using the BCA method. There was a statistical significant difference in the total protein concentration between the two different (cell and lysat fraction) described ways. Furthermore, it is a fast and easy method to extract proteins from ear wax. The benefit of the described method and the field of application will be discussed.

Malassezia otitis externa in the dog: the effect of heat-fixing otic exudate for cytological analysis.

This study was conducted on 32 dogs with Malassezia otitis externa to determine the effect of heat-fixing otic exudate on cytological analysis. Malassezia infection was confirmed by cytological examination of otic exudate. Otic discharge collected with cotton swabs was then rolled onto glass slides. One slide per dog was heat-fixed prior to staining; the other slide was not heat-fixed. The number of yeast in 10 oil-immersion fields (1000 x magnification) was counted for both slides from each dog. Heat-fixing did not systematically cause either increased or decreased numbers of Malassezia on cytology of otic exudate.

Comparison of 4 fixation and staining methods for the cytologic evaluation of ear canals with clinical evidence of ceruminous otitis externa.

BACKGROUND: Swab cytology of ear canals is one of the most useful and rapid methods to assess the presence of external ear infections. Smears are generally stained with rapid Romanowsky-type stains, with or without prior heat fixation. OBJECTIVES: The aim of this study was to compare 4 different methods of fixation and staining of ear swab cytology samples from dogs. METHODS: Eight dogs with otitis externa were selected from a dermatology referral population. A cotton swab was used to obtain ceruminous material from 12 ear canals. Four smears of each swab were prepared on glass slides (randomly identified as A, B, C, or D) and air-dried for cytologic examination. Samples marked A were stained with Dip Quick (Jorgensen Laboratories Inc, Loveland, CO, USA) after heat fixation; samples marked B were stained without heat fixation; samples marked C were heat-fixed and dipped only in the counterstain (the blue reagent) of Dip Quick; and samples marked D were dipped only in the counterstain, without heat fixation. Ten high-power fields (hpf; X100 oil immersion objective) in each slide were evaluated by 2 observers, and total numbers of keratinocytes, yeast, bacteria, and neutrophils were counted. Statistical comparison was performed using an ANOVA model applied after verifying the normal distribution of the data, and using nonparametric sign tests and Wilcoxon signed rank tests. RESULTS: No statistically significant differences were observed in the numbers of keratinocytes, yeast, bacteria, or neutrophils among the 4 staining methods (P > .05), although significant interobserver differences were found. CONCLUSION: We conclude that heat fixation does not improve the quality of ceruminous ear swab samples for cytologic evaluation, and propose a 1-step dip in the blue reagent alone as a rapid method of staining samples from canine ear canals.

Cytology of subcutaneous glandular tissues.

This article deals with the cytologic appearance of various glandular tissues located in the subcutaneous tissues. Normal cytologic features are described. In addition, inflammatory, infectious, neoplastic, and hyperplastic changes are discussed. Most of these features are depicted in the 60+ photomicrographs that are distributed throughout the article. Many of the changes are similar in the glands, and it is usually possible to differentiate the gland of origin based on cytologic appearances. Subcutaneous neoplasms that are not associated with a subcutaneous gland, and lymph node cytology are not covered in this article but are addressed elsewhere.

Gross cystic disease fluid protein in nipple aspirates of breast fluid of Asian and non-Asian women.

Gross cystic disease fluid protein 15 (GCDFP-15) is universally present in the apocrine metaplastic epithelium of cystic breast disease and breast cancer, but it is rarely found in normal breast epithelium. Therefore GCDFP-15 detected in nipple aspirates of breast fluid (NAF) could serve as a biochemical marker of the presence and possibly extent of apocrine metaplasia within the breast. GCDFP-15 levels were measured in NAF from 37 Asian and 78 non-Asian women using radioimmunoassay. GCDFP-15 (range, 0-81,643 micrograms/ml) was found in the NAF of all but 1 woman and was highly correlated between right and left breasts. Mean concentrations of GCDFP-15 were significantly lower in NAF from Asian compared with non-Asian women. Markedly reduced levels of GCDFP-15 were found in the 17 women who had been parous in the previous 2 years. In women not parous within the prior 2 years, no relationship was found between GCDFP-15 levels and age, weight, age at menarche, first-degree family history of breast cancer, parity, oral contraceptive use, or smoking history. High concentrations of GCDFP-15 were found in the NAF of women with a history of a benign breast biopsy. Because similarly high levels of GCDFP-15 were found in NAF in over 40% of women without a history of benign breast biopsy, and because GCDFP-15 in the breast is produced only by apocrine metaplastic epithelium, we infer that the breasts of these women likely contain a significant degree of apocrine metaplasia.(ABSTRACT TRUNCATED AT 250 WORDS)

Swirls, wrinkles and the whole ball of wax (the source of keratin in cerumen).

Recent work carried out at the Ear Pathology Research Laboratory has demonstrated that cerumen plugs consist of a large amount of keratin debris. In this study the site of origin of this keratin was investigated. This was done by histologically examining these cerumen plugs without disturbing the natural relationship between the plug and the surrounding epithelium. We have clearly demonstrated that a cerumen plug consists of keratin arising from the migratory epithelium of the deep external auditory canal and epithelium of the superficial external auditory canal.

The mechanism of ceruminolysis.

In a previous study comparing the efficacy of a selection of commonly used ceruminolytics, the authors noted that aqueous-based preparations, and in particular solutions of sodium bicarbonate, were more effective in disintegrating cerumen than most organic-based preparations. In that study, the authors also observed that not only had the wax truly disintegrated following exposure to the aqueous-based preparations, but also that a marked degree of swelling of the wax spheres had occurred with these preparations. In this paper the mechanism of ceruminolysis was investigated by means of a number of commonly available histological techniques. Our findings show that desquamated sheets of corneocytes are the major constituent of cerumen plugs and provide the structural framework of the wax bolus. Ceruminolytics work by hydrating the keratin cells of these sheets of desquamated stratum corneum and subsequently inducing keratolysis, with disintegration of the wax.

Morphological differences between secretory cells of wet and dry types of human ceruminous glands.

Morphological differences between secretory cells of the wet and dry types of human ceruminous glands were examined. The heights of secretory cells varied from tall and medium to low in both wet- and dry-type glands. The two gland types differed in morphologic features of the tall cells and the cells of medium height. The Golgi apparatus was well developed in the tall cells and fairly well developed in the cells of medium height in the wet-type gland, whereas it was generally small in the corresponding cells of the dry type. Light granules were abundant in the tall cells and in the cells of medium height in the wet-type gland, whereas light granules were rare in these cells in the dry-type gland. Furthermore, the light granules in the wet-type gland cells were observed in close relation to a well-developed Golgi apparatus, and sometimes showed a morphologic appearance suggesting exocytosis. Apical protrusions, probably related to apocrine secretion, were generally large and round and bore "microvilli and light granules" or "very few microvilli and no light granules" in the tall cells of the wet-type gland. However, the protrusions of the tall cells of the dry-type gland were generally large and slender and possessed no microvilli and no granules. The protrusions were not observed in the cells of medium height or in low cells in either type of gland. The results show that eccrine secretion characterizes the wet-type gland, but it is not clearly evident in the dry-type gland. This differences may be related to differences in composition between the wet and dry cerumens.

Transmission and scanning electron microscopy of the human ceruminous apocrine gland. I. Secretory glandular cells.

The secretory portion of the apocrine sweat gland of the human external auditory meatus which is also called the ceruminous gland was observed by transmission and scanning electron microscopy. The secretory glandular cells contain a well developed smooth endoplasmic reticulum and Golgi apparatus. Cisternae of rough endoplasmic reticulum are often closely applied to large round mitochondria. These large mitochondria have no relationship to the secretory granules. Near the concave surface of the Golgi lamellae several tubules can be found. In these golgi-associated tubules a dark substance may accumulate to form specific large dense granules. Many less dense droplets or vacuoles may appear in these dark prosecretory granules and become liberated from their surface. Vacuoles formed in this manner then migrate to the apical cell surface and often discharge their contents into the gland lumen by the mechanism of exocytosis. Also, some of these vacuoles may be released into the lumen by the pinching off of small protrusions of cytoplasm, that is, they are released by the so-called apocrine secretory mechanism. Acid phosphatase activity was demonstrated not only in the dark prosecretory granules but also in clear vacuoles situated at the apical end of the cell and in the gland lumen. Such a histochemical finding may indicate that the secretory substance of the apocrine sweat gland may contain hydrolytic enzymes derived from lysosomes, which are the prosecretory granules of this gland, and these enzymes may play a role in dissolution and break down of the material extruded into the lumen by apocrine secretion. Apocrine secretory process of various sizes were observed on the luminal surface with the scanning electron microscope.

Transmission and scanning electron microscopy of the human ceruminous apocrine gland. II. Myoepithelial cells.

The myoepithelial cells of the human ceruminous apocrine gland were observed with transmission and scanning electron microscopes. The myoepithelial cells are long fibrous cells about 100-150 mum in length and 3-5 mum in width. They are arranged in parallel with each other, and their long axes are parallel to that of the secretory tubule itself. The tips of cells are often sharply pointed and their lateral tapering surface may be contiguous with adjacent cells forming a side-by-side contact, while other cells may have a blunt tip which is conjuncted with a similar tip of the next cell forming an end-to-end junction. The myoepithelial cells are joined to each other by desmosomes and there are also desmosomes at their junction with secretory cells. The outer surface of the cell abutting on the basal lamina has some exaggerated densities which are undoubtedly identical to the hemidesmosomes of epidermal cells. There are well developed foldings in the plasma membrane of the secretory cells, but the surface of the myoepithelial cells has very few foldings and projections. The relative shortage of intercellular attachment devices between the secretory and myeopithelial cells makes it easy to peel off the secretory cells to disclose the myoepithelium, a useful feature of scanning electron microscopy. The nucleus-containing part of the cell protrudes slightly upward and invades the secretory epithelium. The cytoplasmic rim surrounding the nucleus contains a small Golgi apparatus and some other organelles. The cytoplasm of the basal half of the cell contains closely packed myofilaments running parallel to the long axis of the cell. There is no definite arrangement of thin and thick myofilaments. Microtubules which often occur in pairs are arranged parallel to the myofilaments.