Mites in pulmonary cytology specimens

Mites were observed in 55 pulmonary specimens examined routinely over a 4.5-yr period. The 55 specimens included 33 spontaneous and 12 aerosol-induced sputum samples, nine bronchial washings, and one fine-needle aspirate. Two specimens contained only fragments of mites; however, in most cases the mites were relatively intact. Many were identified as Tyrophagus sp, but a few were definitely not Tyrophagus sp. Although only a single mite was noted in most instances, eight specimens contained more than one, with the maximum being six. No patient was found to have a mite in more than one specimen. Mite eggs were found in five of the specimens containing mites. One specimen contained adults and a larval form. Mites appeared to be surrounded by acute inflammatory cells in 13 cases. Specimens were processed by the Saccomanno blending technique with smears prepared by pipetting a portion directly onto a slide or by utilizing a cytocentrifuge. No mites were found in nonpulmonary specimens. Both a seasonal variation and an interobserver variability were found. Mites often resemble specimen contaminants, such as food, and may be overlooked on routine screening. Mites are known to represent or produce a respiratory allergen, but the significance of their presence in pulmonary cytology specimens has not been determined.     

Doing more with less: New markers for sarcoma diagnosis and their applicability to cytology specimens

Sarcomas are a rare and extremely diverse set of neoplasms that are often a challenge to diagnose for pathologists. For a number of reasons, primary diagnosis of soft tissue neoplasms is increasingly being performed on small biopsy specimens including fine needle aspiration (FNA) and core needle biopsy (CNB). In the last several years, there has been a significant increase in our understanding of the molecular pathogenesis of this group of tumors. New insights into the genetic mechanisms of tumor formation have been exploited to create a new generation of diagnostic markers that are accessible to most laboratories. This review describes a number of new ancillary markers, and how they can facilitate accurate diagnosis of soft tissue neoplasms on FNA/CNB. Diagn. Cytopathol. 2016;44:351–360. © 2016 Wiley Periodicals, Inc.     

Laboratory assessment of physical and chemical methods of preserving urine specimens.

Three methods of preserving simulated specimens of urine were studied with six test strains of bacteria. Viable counts were measured by a surface viable count and by the filter-paper-strip method during a holding period of 72 hours. Refrigeration at approximately 4 degrees C was effective and reliable. Boric acid (1-8%) at room temperature was toxic for the strain of Escherichia coli at a density of 10(7) cfu/ml but this may not be significant at the higher concentration of bacterial cells often found in clinical specimens. NaCl-polyvinylpyrrolidone (PVP) solutions containing PVP of mol. wt 44 000 or 700 000 were not effective; they were toxic for the Gram-negative strains and did not retard the growth of Micrococcus subgroup 3. The two methods of measuring viable counts were compared for specimens held under different conditions; the specificity of the filter-paper-strip method was high but the sensitivity was low when many of the specimens contained approximately 10(5) cfu/ml.     

EGFR expression as an ancillary tool for diagnosing lung cancer in cytology specimens.

Lung cancer evolves in a multistep process, and its early detection portends a better prognosis. Bronchial washings/brushings and fine-needle aspirations are often used as early screening and cytological diagnosis of lung cancer. In some cases, it is difficult to differentiate morphologically malignant from reactive cells. Epidermal growth factor receptor (EGFR) is a transmembrane receptor overexpressed in high percentage lung cancers, and contributes to tumor growth. Assessing EGFR expression levels by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) may provide critical information of tumor marker abnormalities, assist in the cytological diagnosis, and stratify patients for EGFR inhibitor therapy. Fifty patients with bronchial washings/brushings or fine-needle aspiration specimens, and corresponding histologically confirmed lung biopsies, were studied for EGFR expression with FISH and IHC. Copy numbers of the EGFR gene locus were analyzed with those of chromosome 7 by FISH. EGFR and FISH results were compared to our FISH data with combined EGFR, c-myc, 5p15.2, and chromosome 6 probes in selected cases. Cell blocks, if available, and tissue biopsy sections were used for EGFR IHC. The intensity of IHC was scored, and quantified. Only balanced aneuploidy of EGFR was identified by FISH. Gene amplification was not detected. The chromosomal abnormalities of EGFR were often accompanied by other chromosomal aneuploidies demonstrated in c-myc (8q24), 5p15.2 or 6p, indicating a general genomic instability. About half of the specimens with confirmed malignancy showed EGFR balanced aneuploidy by FISH, and gene copy number was not coupled with protein expression in many cases. The benign or reactive cytology specimens confirmed by biopsies had high specificity by FISH (96%) and IHC (88%). FISH and IHC analysis of EGFR, possibly along with other tumor markers, may be a useful ancillary tool to classify difficult cytology cases and inform clinicians arranging targeted chemotherapy.     

A novel filtration-based processing method of liquid cytology specimens for human papillomavirus DNA testing by hybrid capture II

We evaluated a more efficient method of processing liquid-based cervical cytology specimens for human papillomavirus (HPV) DNA testing by Hybrid Capture II (HCII). Aliquots were made from 701 specimens in the following sequence: 4.0, 2.0, 1.0, 0.5, and 1.5 mL. The 4.0-mL aliquot was processed by the standard method (STP), and half of the processed material was tested by HCII. Other aliquots were processed with a new, filtration-based processing method (NPM). The 2.0-mL NPM aliquot had HCII test performance most similar to the STP, ie, similar HCII positivity (P = .4) and good test agreement (kappa = 0.85, 95% confidence interval [CI], 0.80-0.89). The 194 cytologic negatives had greater positivity by STP (P = .04) compared with the 2.0-mL aliquot processed by NPM; between-method agreement was modest (kappa = 0.54, 95% CI, 0.36-0.72). A lower positive cut point for the 2.0-mL NPM aliquot partially abrogated this minor difference. In 241 specimens diagnosed as low-grade and 31 as high-grade squamous intraepithelial lesions, there were no significant differences in HPVpositivity (>85% and 90%, respectively) between STP and NPM. NPM reduces specimen handling and decreases total testing time by approximately 33% without significant losses in HCII test performance.     

Comparison of immunomarkers for the identification of adrenocortical cells in cytology specimens

We studied the immunoreactivity of three antibodies--A103, calretinin, and inhibin alpha in destained Papanicolaou (Pap) smears and cell-blocks of 40 fine-needle aspiration biopsy cases of adrenocortical lesions (35 cases of hyperplasia/adenoma and 5 cases of carcinoma). Five cases of carcinoma (4) and melanoma (1) metastases to the adrenal gland and five cases of renal-cell carcinoma were also included for comparison. In benign adrenocortical lesions, A103 staining was noted in 82% of the destained Pap smears and in 92% of cell-blocks. In malignant adrenocortical lesions, A103 staining was noted in 50% of the destained Pap smears and in 80% of cell-blocks. In comparison, calretinin staining was noted in 6% and 50% of destained smears and in 78% and 60% of the cell-blocks of benign and malignant adrenocortical lesions. Inhibin alpha was not positive in any of the smears and showed the lowest level of positivity in the cell-block sections, namely in 11% of the benign lesions and 25% of the malignant lesions. The sensitivity of A103 was 90% on cell-blocks and 74% on smears, that of calretinin 75% on cell-blocks and 11% on smears, and that of inhibin alpha, 13% on cell-blocks alone. The specificity of A103 was lower than the other two makers, 90% vs. 100% because of positivity in metastatic melanoma in the adrenal gland.Our data show A103 to be the immunomarker with the highest sensitivity for identifying cells of adrenocortical origin in destained Pap's smears and cell-block sections with, however, a lower specificity when compared with calretinin and inhibin alpha. Calretinin is comparable in sensitivity with A103 on cell-block sections alone and not on smears. The results of this study suggest that if metastatic melanoma in adrenal gland is not a consideration then A103 is the marker of choice for identifying cells of adrenocortical origin in the limited material available for diagnostic purposes in cytology specimens.     

MALDI imaging as a specific diagnostic tool for routine cervical cytology specimens

Cervical squamous cell carcinoma (SCC) is among the most common malignancies in women worldwide. In developed countries routine cytology screening has dramatically reduced SCC mortality within the last three decades. High risk (HR) human papilloma virus (HPV) infection is the main causal factor in nearly 100% of invasive SCCs, in most cases of low grade squamous intraepithelial lesion (LSIL) and in nearly all cases of high grade squamous intraepithelial lesion (HSIL). Detection of HR-HPV DNA has been extensively evaluated for the triage of patients with low grade cytological abnormalities in order to identify those at greatest risk for underlying or developing HSIL or SCC. However, the vast majority of HR-HPV-positive precursor lesions will not progress to invasive cancer. A variety of other screening tools are available which aim to stratify clinically significant HPV infections and cytological alterations. Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry is a promising technology to assist in this endeavor. It delivers accurate mass spectrometric information of the sample's proteins and enables the visualization of the spatial distribution of protein expression profiles in correlation with histological features. In this study, 18 samples with Pap IIID or higher (>LSIL) and 14 samples with Pap I-II (WNL) were analyzed by MALDI imaging mass spectrometry (IMS). A genetic algorithm was applied to classify spectra resulting in an overall cross validation, sensitivity for Pap IIID and Pap?I-II of 83.7%, 88.9% and 78.6%, respectively. As this IMS based approach allows for unbiased and automated classifiction of cytological samples it appears to be a promising tool for stratification of cervical Pap smears.     

The proliferative cell fraction in cytology specimens. A study of human esophageal carcinoma.

Immunostaining of cell cycle-related antigens, especially Ki-67, DNA polymerase alpha, and proliferating cell nuclear antigen, has become an important method to assess the proliferative activity of tumors. These three nuclear antigens were studied by immunohistochemical analysis of cytologic smears. These smears were obtained by scraping the cut surface of 10 cases of esophageal squamous cell carcinoma and were fixed and prepared by different methods. The results were compared with those of tissue sections to apply the immunocytochemical findings of these antigens to cytology specimens. Smears that were placed on Denhardt- or Neoprene-coated slides and subsequently fixed in 4% paraformaldehyde and methanol exhibited the best cell adherence to the slides, had minimal loss of antigenicity, and had good preservation of cell morphologic features for all three antigens examined. The percentage of positive tumor cells in the cytology smear was generally in good agreement with that in the tissue section. For these three antigens, proliferating cell nuclear antigen demonstrated a much higher percentage of positive cells than either Ki-67 or DNA polymerase alpha, in both the smears and the tissue sections. In summary, Ki-67, DNA polymerase alpha, and proliferating cell nuclear antigen can be immunolocalized successfully in cytology smears and may become another parameter to assess the proliferative activity of tumors in the field of diagnostic pathology.     

Detection of malignancy in cytology specimens using spectral-spatial analysis

Despite low sensitivity (around 60%), cytomorphologic examination of urine specimens represents the standard procedure in the diagnosis and follow-up of bladder cancer. Although color is information-rich, morphologic diagnoses are rendered almost exclusively on the basis of spatial information. We hypothesized that quantitative assessment of color (more precisely, of spectral properties) using liquid crystal-based spectral fractionation, combined with genetic algorithm-based spatial analysis, can improve the accuracy of traditional cytologic examination. Images of various cytological specimens were collected every 10 nm from 400 to 700 nm to create an image stack. The resulting data sets were analyzed using the Los Alamos-developed GENetic Imagery Exploitation (GENIE) package, a hybrid genetic algorithm that segments (classifies) images using automatically 'learned' spatio-spectral features. In an evolutionary fashion, GENIE generates a series of algorithms or 'chromosomes', keeping the one with best fitness with respect to a user-defined training set. First, we tested the system to determine if it could recognize malignant cells using artificial cytology specimens constructed to completely avoid the requirement for human interpretation. GENIE was able to differentiate malignant from benign cells and to estimate their relative proportions in controlled mixtures. We then tested the system on routine cytology specimens. When targeted to detect malignant urothelial cells in cytology specimens, GENIE showed a combined sensitivity and specificity of 85 and 95%, in samples drawn from two separate institutions over a span of 4 years. When trained on cases initially diagnosed as 'atypical' but with unequivocal follow-up by biopsy, surgical specimen or cytology, GENIE showed efficiency superior to the cytopathologist with respect to predicting the follow-up result in a cohort of 85 cases. We believe that, in future, this type of methodology could be used as an ancillary test in cytopathology, in a manner analogous to immunostaining, in those situations when a definitive diagnosis cannot be rendered based solely on the morphology.     

Gelatin foam cell blocks made from cytology fluid specimens

This report describes a simple method of preparing cell blocks from fluids submitted for cytology, using croutons of gelatin foam surgical dressing material.     

Rapid method for detecting beta-lactamase producing bacteria in clinical specimens.

The use of liquid media to detect the production of beta-lactamase by beta-lactamase producing organisms has been compared with the conventional method of inoculation on to agar media. Pharyngeal cultures were obtained from 162 children treated with penicillin for acute tonsillitis. beta-lactamase producing organisms were detected within 72 h in 80 (49%) of the specimens inoculated on to agar media, while beta-lactamase production was found in 76 (47%) of the specimens after their incubation in liquid media for 24 h. Twenty one of the cultures were positive only after anaerobic incubation while in liquid media while nine were positive only after aerobic incubation. Incubation in liquid media enabled detection of beta-lactamase activity in 53 of the 76 (70%) specimens within 12 h.     

Comparison of Cyto-Shuttle® and cytocentrifuge as processing methods for nongynecologic cytology specimens

The Cyto-Shuttle® (Cancer Diagnostics, Inc., Fairfax, VA) monolayer preparation method was compared to our routine cytocentrifuge method in 129 fluid cytology cases. A single sample from each case was split and prepared by each method. The Cyto-Shuttle preparation was superior to the cytocentrifuge preparation in 51% of cases, equal to it in 38%, and inferior to it in 11%: bronchial wash/lavage (45 cases), 38%, 49%, 13%; body cavity fluid (39 cases), 72%, 15%, 13%; urine (18 cases), 56%, 44%, 0%; peritoneal washing (16 cases), 44%, 44%, 12%; and miscellaneous (11 cases), 36%, 55%, 9%. Cyto-Shuttle preparations were superior due to decreased background and increased number of cells per slide; fixation and morphology were generally equivalent to cytocentrifuge preparations. Diagn Cytopathol 1996;14:305–309. © 1996 Wiley-Liss, Inc.     

A transport method for swab specimens submitted for aerobic and anaerobic bacteriology.

The need for separate swab transport methods for aerobes and anaerobes may result in inadequate transport of specimens for anaerobic bacteriology. Most microbiology laboratories in Australia rely on Stuart's transport medium to protect anaerobic bacteria. This paper presents a new, simple transport medium (Transport Deep) suitable for sue with aerobes and anaerobes. Comparative evaluations demonstrate that Transport Deep is as good as Stuart's medium for the maintenance of fastidious bacteria and is far superior for the protection of even extremely oxygen-sensitive anaerobes. This medium has been used successfully in a large Sydney hospital for more than a year. It is proposed that Transport Deep be used on a routine basis for all swab specimens.