肺癌细胞学亚型巴氏染色的计算机图像色度学定量分析

用计算机图像处理技术,对痰涂片中巴氏染色的角化性鳞癌细胞（ＫＳＣＣ）、非角化性鳞癌细胞（ＮＫＳＣＣ）和腺癌细胞（ＡＣＣ）胞浆进行了色度学定量分析。测试参数为红（Ｒ）、绿（Ｇ）、蓝（Ｂ）三基包含量和Ｒ、Ｇ、Ｂ的三色系数,同时还测算了胞浆的色度、饱和度、亮度和灰度,并进行了逐步判别分析。结果表明：ＫＳＣＣ的Ｒ、Ｇ、Ｂ及其三色系数、色度、饱和度与ＮＫＳＣＣ和ＡＣＣ相比差异非常显著;三基色的改变较灰度敏感．用三色系数、色度、饱和度对ＫＳＣＣ与ＮＫＳＣＣ、ＫＳＣＣ与ＡＣＣ进行计算机判别,准确度可达９５．３％和９７．１％。这些结果提示：三基色、三色系数及色度、饱和度分析对判别ＫＳＣＣ与ＮＫＳＣＣ、ＫＳＣＣ与ＡＣＣ有重要价值。对有关问题进行了讨论。     

Quantitative and qualitative analysis of stain color using RGB computer color specification

OBJECTIVE: To establish standardized Papanicolaou stain for cytology using RGB color specification. This new method was formerly used in DTP software application for computer color specification. STUDY DESIGN: RGB color specification was taken from a color film, optical constituents of which were made into computer software. Cell samples used in this study were from 100 sputum specimens stained with Papanicolaou stain. We analyzed the color tone of the cytoplasm of squamous cells in the smear. RESULTS: The R and B value of eosinophilic cells were demonstrated statistically by different values between normal, borderline atypical and malignant squamous cells. G and B values of light green-philic cells demonstrated a statistical difference between normal, borderline atypical and malignant squamous cells. No significant differences were found in RGB value between normal, borderline atypical and malignant squamous orangeophilic cells. CONCLUSION: Using our own method of analyzing Papanicolaou-stained sputum, a new quantitative and qualitative analysis of stain color for standardized Papanicolaou stain was introduced.     

Colorimetry for the stain technologist. IV. Analysis of the components of color difference

Total color differences have been calculated for various pairs of stained microscopic substrates. The latter include azure B/eosin stained blood cells and Papanicolaou stained cells from the uterine cervix. Both the CIE L*u*v* and L*a*b* color spaces have been used. Total color differences have been analyzed in terms of lightness, hue and chroma components. Various discrepancies have been noted among these components, especially the chroma difference, for the two spaces. It is concluded that current color-difference formulae are less than perfect, although they can provide much useful information.     

Application of Ultrafast Papanicolaou stain to body fluid cytology

OBJECTIVE: To investigate the applicability of the Ultrafast Papanicolaou stain to the cytology of fluids and to compare it with other methods. STUDY DESIGN: Over a 30-month period, 528 unfixed fluids (462 serous effusions, 48 pelvic washings, 16 cyst fluids and 2 bile duct drain fluids) were mixed thoroughly and centrifuged. Two Swedish-style air-dried smears were made and stained with Diff-Quik (Mercedes Medical, Inc., Sarasota, Florida, U.S.A.) and Ultrafast Papanicolaou stain (Richard Allan Scientific, Kalamazoo, Michigan, U.S.A.), and the remaining sediment was fixed in CytoRich Red (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.), centrifuged onto a 17.5-mm circle with a Hettich cytocentrifuge and stained by the Papanicolaou method. RESULTS: For the 115 malignant fluids, Ultrafast Papanicolaou stain was the preferred method in the 94 non-hematopoietic malignant fluids, Diff-Quik was the preferred method in the 9 hematopoietic malignancies, and CytoRich Red was the preferred preparation in 8 bloody effusions containing rare cancer cells and 4 malignant pelvic washings. The diagnostic turnaround time of smears stained by Ultrafast Papanicolaou stain was < 15 minutes, fast enough for intraoperative consultations. CONCLUSION: It seems that Ultrafast Papanicolaou stain improves the resolution of cytoplasmic and nuclear details of nonhematopoietic cells in body fluids. However, to detect cancer in all types of fluids, Diff-Quik and CytoRich preparations are also required. We now examine three slides per fluid sample, one slide by each of the three techniques.     

Color image analysis of cervical neoplasia using RGB computer color specification

OBJECTIVE: To establish quantitative color image analysis for cytology, red, green and blue (RGB) color specification was applied to Papanicolaou-stained cervical smears. STUDY DESIGN: Cell samples used in this study was those from 300 cervical specimens. We analyzed the color tone of nuclei and cytoplasm of the squamous cells in the cervical smear by means of computer image analysis. RESULTS: Papanicolaou stained nuclei displayed basophilic blue to purple. When they were hyperchromatic and deeply stained, B and G values decreased in value. The RGB values of cytoplasm and nuclei decreased significantly (P < .01) as their degree of cellular atypia increased. CONCLUSION: Using RGB color specification to analyze Papanicolaou-stained cervical smears, a significant difference was perceived in the nucleus and cytoplasm between different groups of squamous cells, from normal, dysplastic and squamous cell carcinoma. These findings may help to establish automated cytology.     

Histological subtypes and characteristic structures of HPV-associated oropharyngeal carcinoma; study with Japanese cases

Background

Human papillomavirus-associated oropharyngeal carcinoma (HPV-OPC) is clinicopathologically distinct entity from the HPV-unassociated one (nHPV-OPC). This study aimed to determine the relationship between histological subtypes of OPC and HPV status for Japanese cases and to identify histological structures of HPV-OPC.

Methods

66 OPC cases were categorized into conventional squamous cell carcinoma (SCC) and the variants. Conventional SCC was subcategorized into keratinizing (KSCC), non-keratinizing (NKSCC), and hybrid SCC (HSCC). HPV status of all cases was determined using p16-immunohistochemistry and HPV-DNA ISH.

Results

Two histological subtypes, NKSCC and HSCC, tended to be HPV-OPC and KSCC tended to be nHPV-OPC with statistical significance. Two histological structures, abrupt keratinization, defined in the text, and comedo-necrosis among non-maturing tumor island, were observed for 58.1% and 38.7% of HPV-OPC, and tended to exist for HPV-OPC with statistical significance.

Conclusions

This study showed the association of NKSCC/HSCC with HPV-OPC in Japanese cases, and two histological structures, abrupt keratinization and comedo-necrosis among non-maturing island, were considered characteristic histological features of HPV-OPC.

Virtual slides

The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1816432541113073.

     

Usefulness of ploidy, AgNOR and immunocytochemistry for differentiating benign and malignant cells in serous effusions

OBJECTIVE: The objective of this study was to establish the value of different markers in differentiating reactive mesothelial cells from metastatic adenocarcinomatous cells in serous effusions (SE). METHODS: Forty-five SE were processed for morphological examination (Papanicolaou stain), assessment of ploidy, AgNOR counting and immunocytochemical assay of carcinoembryonic antigen (CEA), epithelial membrane antigens (EMA), Ber-EP4 and Leu-M1. Ploidy was established in an image-analyser in smears stained by the Feulgen stain method. AgNOR dots were counted in the smears stained with the silver nitrate assay for non-histone proteins present in the nucleolar organizer region. CEA, EMA, Ber-EP4 and Leu-M1 were evaluated by immunocytochemistry using the streptavidin-biotin complex method. RESULTS: All the smears with positive cytology were aneuploid. Three false negatives by morphological studies were aneuploid, with AgNOR values in two of them corresponding to the neoplastic group. CEA and Leu-M1 showed a low specificity; EMA and Ber-EP4 showed moderate sensitivity. CONCLUSIONS: The assessment of ploidy and the study of AgNOR were better methods than immunocytochemistry for distinguishing between reactive mesothelial cells and adenocarcinomatous cells in serous fluid.     

The use of p63 as an effective immunomarker in the diagnosis of pulmonary squamous cell carcinomas on de-stained bronchial lavage cytological smears

M. Uke, B. Rekhi, D. Ajit and N. A. Jambhekar
The use of p63 as an effective immunomarker in the diagnosis of pulmonary squamous cell carcinomas on de‐stained bronchial lavage cytological smears Objectives: A diagnosis in pulmonary onco‐cytopathology primarily necessitates distinguishing small cell carcinoma (SCLC) from non‐small cell carcinoma (NSCLC), which includes squamous cell carcinoma and adenocarcinoma. Lately, p63 antibody has been used for distinguishing squamous cell carcinoma from SCLC and adenocarcinoma. We present an analysis of p63 expression in cytological smears from 100 bronchial lavage specimens comprising 51 cases of SCLC and 49 cases of NSCLC. Methods: A single Papanicolaou‐stained conventional smear was de‐stained and re‐fixed with cold acetone and methanol for immunocytochemical staining with p63 antibody. Staining results were graded as 0 (nil), 1+ (focal), 2+ (moderate, diffuse) and 3+ (strong, diffuse). Results: Out of 100 cases, 21 were cytologically diagnosed as squamous cell carcinoma. Twenty of these showed 2+ or 3+ p63 positivity, whereas one, which was adenocarcinoma on histology, showed 1+ staining. Of seven cases cytologically diagnosed as adenocarcinoma, six showed no p63 staining, whereas one, which was squamous cell carcinoma on histology, showed 1+ staining. All 48 cases cytologically diagnosed as SCLC were confirmed as such on histology and showed no p63 staining. Four cases were cytologically designated as poorly differentiated carcinomas, of which three showed no p63 staining and one showed 3+ staining. The former three were found to be SCLC on histology while the latter was squamous cell carcinoma. The remaining 20 cases were cytologically designated as NSCLC. Of these, eight showed no p63 staining, whereas 10 showed 1+ and two showed 2+ staining. The former eight were adenocarcinoma on histology and the latter two were squamous cell carcinoma. The 10 cases that showed 1+ p63 staining were adenocarcinomas (n = 5), squamous cell carcinoma (n = 4) and NSCLC, not otherwise specified (n = 1). Positive staining was seen in normal basal cells, which acted as an internal control. Overall sensitivity of p63 for squamous cell carcinoma was 100% and specificity was 90.4%. Conclusions: p63 immunostaining on processed cytology smears can be used to help identify squamous cell carcinoma. Its diffuse expression was specific for squamous cell carcinoma while focal staining was also seen in adenocarcinoma.     

Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.     

Stains for the Primary Wall of the Cotton Fiber

Several methods are described for distinguishing between the primary wall of the cotton fiber and other fiber components, such as the lumen and the secondary wall. The primary wall, a membrane less than 0.5 μ thick covering the entire fiber, has been stained while still attached to the fiber as well as after it has been mechanically stripped from the fiber. The stains include aqueous or alcoholic solutions of ruthenium red, methylene blue chloride, Nile blue sulfate, oil red, Sudan black B, iodine, and Simons' stain. Various concentrations of sodium hydroxide, cupri-ethylenediamine hydroxide, or sulfuric acid have been used to enhance color changes and to cause cellulosic swelling. Fibers that have been stained with Simons' stain and then swelled with dilute cupri-ethylenediamine hydroxide have shown the greatest color differences between the primary wall and the lumen.     

Usefulness of AgNOR technique and CEA expression in atypical metaplastic cells from cervical smears

OBJECTIVE: To evaluate the proliferating capacity of atypical metaplastic cells in cervical smears by AgNOR technique and image analysis and investigate the probable relation with squamous intraepithelial lesions (SIL) using immunocytochemical assay for carcinoembryonic antigen (CEA). STUDY DESIGN: Eight cervical smears were stained with Papanicolaou stain for diagnosis of atypical metaplastic cells. After removal of the stain the smears were processed with a silver colloidal solution and the AgNOR area determined by image cytometry. Differences in the mean AgNOR protein area between reactive metaplastic cells and controls were tested by Student's t test. The CEA was investigated by immunocytochemical staining in smears with atypical metaplastic cells and smears from high-grade squamous intraepithelial lesions (HSIL). RESULTS: The mean AgNOR areas from atypical metaplastic cells (4.55, 6.66, 4.68, 5.30, 4.97, 6.20, 6.28, and 7.35) were significantly greater those that of intermediate metaplastic cells and cells from low-grade squamous intraepithelial lesions (LSIL) (0.77, 0.99, and 0.82, respectively). The atypical metaplastic cells showed values of mean AgNOR area intermediate between that of basal cells (3.28) and HSIL cells (7.73) or neoplastic cells (16.12). The CEA was strongly expressed by the atypical metaplastic cells. CONCLUSION: The expression of CEA in the atypical metaplastic cells underlies the probable relation to SIL. Although the organizer region areas raised high values, it would be necessary to use a greater number of cases to define whether the AgNOR area is indicative of the proliferative activity of the atypical metaplastic cells.     

Estimation of the chlorophyll content of micropropagated potato plants using RGB based image analysis

A method has been developed for rapid and non invasive determination of chlorophyll content of leaves of micropropagated potato plants using RGB based image analysis. Among the trichomatic colors, R and G negatively correlated with the chlorophyll content, while a positive correlation was observed with B chromate. Compared to mean brightness value, the use of mean brightness ratio considerably improved the relationship of the tricolors with chlorophyll content. The brightness values and ratios of the primary colors are modeled as linear correlation functions for chlorophyll content. A significant correlation was observed between the model predicted chlorophyll content with the chlorophyll content measured by chlorophyll content meter. Spectral properties such as luminosity and saturation were also found to be negatively correlated with the chlorophyll content. The relationship was improved by combining the mean brightness ratio at B band region with luminosity. The potential of the imaging system in micropropagation has been discussed.     

Sialadenitis with crystalloid formation. Fine needle aspiration cytodiagnosis of 15 cases

OBJECTIVE: To assess the utility of fine needle aspiration cytology in the diagnosis of sialadenitis with crystalloid formation. STUDY DESIGN: In 15 cases, salivary gland masses were aspirated using a disposable, 20-mL syringe and 25-gauge needles, maintaining negative pressure. Smears routinely were air dried and stained by Diff-Quik (Dade Behring AG, Düdingen, Germany). Occasionally smears were fixed in alcohol and stained by the Papanicolaou method. RESULTS: The smears showed large numbers of non-birefringent crystalloids of varying sizes and shapes. The crystalloids stained deep blue with Diff-Quik and bright orange with Papanicolaou stain. Multinucleated histiocytes, neutrophilic leukocytes and benign salivary gland parenchyma were found, also. CONCLUSION: Fine needle aspiration cytology provides an accurate diagnosis of sialadenitis with crystalloids and is useful for avoiding unnecessary surgery.     

C. I. Acid Red 52: A New Stain for Cells of Granulocytic Origin

Using the xanthene dye C.I. acid red 52 (CI. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52- In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

Keratinized squamous cells in fine needle aspiration of the brain. Cytopathologic correlates and differential diagnosis

OBJECTIVE: To analyze the differential diagnosis when keratinized squamous cells are found in a brain aspirate. STUDY DESIGN: Twenty cases of brain aspirates with keratinized squamous cells were retrieved (1982-2001). Diagnoses included craniopharyngioma (CP) (n = 11), metastatic squamous cell carcinoma (SCC) (n = 5), epidermoid cyst (EC) (n = 3) and Rathke cleft cyst (RCC) (n = 1). Aspirates were obtained under stereotactic radiologic (CT) guidance. Smears were stained with Diff-Quik or Papanicolaou stain, and cell block sections were stained with hematoxylin and eosin. Radiologic and histopathologic correlation with subsequent resection specimens was performed in selected cases. RESULTS: CP showed cellular smears with numerous keratinized squamous cells in a background of degenerated cellular and keratinaceous debris. Also noted were clusters of anucleate squamous cells, multinucleated giant cells, histiocytes, calcified debris and characteristic fragments of basaloid epithelial cells. Metastatic SCC showed single cells and tissue fragments of markedly atypical and focally keratinized cells with enlarged, hyperchromatic nuclei; prominent pleomorphism in a background of necrotic cellular debris and acute inflammatory exudate. EC showed numerous isolated keratinized squamous cells often with prominent keratohyaline granules and occasional parakeratotic cells in a relatively clean background. RCC showed single cells and aggregates of benign-appearing squamous cells admixed with numerous anucleate squames and hemosiderin-laden macrophages. Glandular-type epithelium was present only rarely. CONCLUSION: Squamous cell-containing lesions in the brain present a spectrum of pathologic entities. Although they all display the common morphologic denominator of keratinizing squamous cells, subtle cytomorphologic differences exist in these lesions, permitting an accurate cytopathologic diagnosis. Clinicardiologic features and anatomic location of the tumor in the brain are additionally helpful.     

Amyloid in cytologic specimens. Differential diagnosis and diagnostic pitfalls.

OBJECTIVE: To describe and illustrate the characteristic features of amyloid in cytologic preparations and point out its diagnostic pitfalls. STUDY DESIGN: Five fine needle aspirates and one bronchial washing that contained amyloid were retrospectively reviewed. The aspirates were obtained from each of the five following sites: lung, occipital lymph node, thyroid gland, proximal humerus and subcutaneous soft tissue. Smears of all of the aspirates were stained with Papanicolaou stain, and in two cases they were also stained with Diff-Quik. Cell block sections were stained with hematoxylin and eosin. Congo red, CD45 and CD20 were used on selected cases. RESULTS: Amyloid appears as either flocculent material or irregularly shaped fragments with scalloped and pointed edges. The amorphous fragments are acellular and frequently associated with connective tissue cells. They stain eosinophilic to cyanophilic with Papanicolaou stain and deep blue with Diff-Quik. In two cases an exuberant giant cell reaction almost obscured the amyloid. In the thyroid aspirate, the amyloid was misinterpreted as colloid. In bronchial washings and lung aspirates, amyloid has to be distinguished from mucus, alveolar proteinosis, chondroid material and corpora amylacea. When circumferentially surrounded by lymphocytes or plasma cells, flocculent amyloid deposits may simulate adenoid cystic carcinoma. CONCLUSION: Amyloid can be easily overlooked or mistaken for other entities with similar staining qualities. Congo red staining can help to confirm the diagnosis.     

Influence of staining on fast automated cell segmentation, feature extraction and cell image analysis

FAZYTAN, a system for fast automated cell segmentation, cell image analysis and extraction of nuclear features, was used to analyze cervical cell images variously stained by the conventional Papanicolaou stain, the new Papanicolaou stain and hematoxylin and thionin only; the last two dyes are used as the nuclear stains in the two versions of the Papanicolaou stain. Other dyes were also tried in cell classification experiments. All cell images in the variously stained samples could be described by the same nuclear features as had been adapted for the discrimination of conventional-Papanicolaou-stained cells. Variances were lower for thionin-stained cells as compared with hematoxylin-stained cells. By application of spectrophotometry, it was confirmed that the spectra of the cytoplasmic counterstains are superimposed on those of the nuclear stains. It appears that a variety of dyes are suitable as cytologic stains for cell classification by the FAZYTAN system, provided that they achieve sufficiently strong nuclear-cytoplasmic contrast by precisely delineating the chromatin texture.     

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions

OBJECTIVE: To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. STUDY DESIGN: Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. RESULTS: Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. CONCLUSION: Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.     

Cytologic diagnosis of acanthamoebic keratitis

A case of acanthamoebic keratitis was identified from a corneal scraping stained with a modified Papanicolaou stain. The characteristic double-walled cyst forms were easily identified. Corneal scraping cytology provides a rapid, noninvasive means of diagnosis and follow-up for this serious disease.