Nuclear Details Research Articles

Rapid staining techniques in cytopathology: a review and comparison of modified protocols for hematoxylin and eosin, Papanicolaou and Romanowsky stains.

The objective of this study was to review and compare rapid protocols for fixation and staining of cytologic smears. We used fresh surgical specimens from dogs and horses to evaluate and modify, if necessary, previously described rapid staining protocols. Slides were wet-fixed, rehydrated or air-dried. Rapid Papanicolaou, hematoxylin and eosin (H&E), and Romanowsky stains were applied, including modification of Diff-Quick stain. The modified rapid staining protocols were simple to use and gave results within 5 minutes that were comparable to those obtained with traditional methods. Advantages of rehydrated vs wet-fixed smears included consistent preparations, a clean background, and equally good or superior nuclear detail.

ThinPrep® vs. conventional smear cytologic preparations in analyzing fine‐needle aspiration specimens from palpable breast masses

Limited data exist concerning the cellular features of the ThinPrep® (Cytyc Corp., Boxborough, MA) technique in the analysis of breast fine-needle aspiration specimens. Therefore, we analyzed a series of 75 surgically excised palpable breast masses and compared ThinPrep and conventional smear fine-needle aspiration preparations. Each mass was aspirated twice. The first sample was used for two alcohol-fixed conventional smears, and the second sample was rinsed into CytoLyt (Cytyc Corp., Boxborough, MA) solution for processing into a ThinPrep slide. The paired slides were separated and independently analyzed for adequacy, overall cellularity, single epithelial cells (absent, rare, moderate, or numerous), epithelial architecture (sheets or three-dimensional clusters), myoepithelial cells and stripped bipolar nuclei (present or absent), and nuclear detail (poor, satisfactory, or excellent). Each sample was classified as negative, negative consistent with fibroadenoma, atypical favoring benign, atypical favoring malignant, or positive for malignant cells. The 75 breast masses included 32 carcinomas and 43 benign lesions. Four conventional smears and one ThinPrep were unsatisfactory. Significantly, more conventional smears were limited by drying artifact (9 vs. 0). ThinPrep aspirates of carcinomas had better nuclear detail (P = 0.03) and greater cellularity (P = 0.05). ThinPrep aspirates of benign masses had greater epithelial cellularity (P = 0.007) and better nuclear detail (P < 0.001), and more specimens had myoepithelial cells (P = 0.007). The ThinPrep interpretation classified 29 of 32 carcinomas (91%) as positive and three as atypical favoring malignant (sensitivity = 100%). The conventional smear interpretation classified 28 of 31 carcinomas (90%) as positive and three as atypical favoring malignant (sensitivity = 100%). The ThinPrep interpretation classified 42 benign lesions as negative (23 cases), negative consistent with fibroadenoma (8 cases), atypical favoring benign (10 cases), and atypical favoring malignant (1 case) (specificity = 74%). The conventional smear interpretation classified 40 benign lesions as negative (25 cases), negative consistent with fibroadenoma (12 cases), and atypical favoring benign (3 cases) (specificity = 93%). ThinPrep was less specific, but the difference was not statistically significant (P = 0.065). In summary, ThinPrep aspirates had greater cellularity and better nuclear detail than conventional smears, and were just as sensitive in identifying the carcinomas. The difference in specificity between the two techniques was not statistically significant (P = 0.065). Diagn. Cytopathol. 1999;21:137–141. © 1999 Wiley-Liss, Inc.

Endocervical Glandular Neoplasia and Its Mimics in ThinPrep® Pap Tests

To document the cytologic features of endocervical adenocarcinoma in situ (AIS) and invasive endocervical adenocarcinoma as observed in ThinPrep slides and to compare these features with those that have been described for conventional smears. Six cases of endocervical AIS and three cases of invasive adenocarcinoma were evaluated with respect to 3 low-power and 14 high-power features. All cases were biopsy proven. Glandular "look-alikes" (tubal metaplasia, n = 3; florid repair, n = 3; sampling of lower uterine segment, n = 1) were also examined. All cases of AIS contained dark groups and sheets at screening power. At higher power, nuclear detail was extremely well visualized. All cases had crowding, continuous depth of focus, variability of nuclear size and shape within groups, irregular nuclear membranes, uniformly stippled chromatin and at least occasional single atypical cells. Only one case lacked nucleoli. Traditional features (strips, feathering, rosettes and mitoses) were observed about as frequently as in conventional smears. Invasive lesions had many of the same features, with relatively more inflammation and lysed blood. Nonneoplastic look-alikes could be distinguished from neoplasms using traditional criteria. In this small study, AIS and invasive endocervical adenocarcinoma maintained the features previously described for conventional smears. Improved visualization of nuclear detail may allow the application of additional criteria, such as irregular nuclear membranes and the more consistent presence of nucleoli, for distinguishing glandular neoplasms from their look-alikes.

Perfect Eyes

Abstract It is extremely difficult to remove artifacts common to eye tissue such as folds, shrinking raisin effect, and parched earth effect. By adjusting our fixation and processing procedures, we attempted to reduce or eliminate these artifacts. We tested the fixatives from various vendors to determine which of them is most compatible with eye tissue and which would eliminate artifacts. We found Pen-Fix (alcoholic formalin; Richard Allen, Richland, Ml) produced optimal fixation (well defined nuclear detail, without bubbling artifact). In similar testing, we found Peel-a-way (Shandon-Lipshaw, Pittsburgh, PA) to be the optimal paraffin for processing and embedding ocular tissues.

Immunohistochemistry of Tartrate-Resistant Acid Phosphatase: Assessment of Steam Heat for Epitope Enhancement and Comparison of B-5 and Formalin Fixation

Tartrate-resistant acid phosphatase (TRAP) is an important cell marker in both hematology and bone pathology. Cytochemical stains for TRAP activity are used to identify leukemic cells of hairy cell leukemia, inflammatory or activated macrophages, and bone resorbing osteoclasts. A monoclonal antibody to TRAP (9C5) was developed for immunohistochemical identification of these and other TRAP-expressing cells in paraffin sections. In our continuing efforts to maximize the sensitivity and specificity of 9C5 for diagnostic and investigational purposes, we conducted studies to compare steam to microwave irradiation for heat induced epitope retrieval (HIER) and to compare formalin fixation to B-5 fixation. We found that a simple and inexpensive household vegetable steamer provided equivalent or superior HIER for demonstration of TRAP with 9C5 in a variety of tissues. We also found no significant differences in immunoreactivity or nuclear detail between formalin and B-5 fixed bone marrow biopsy sections. These findings may also apply to other antigens of diagnostic importance. We concluded that the high quality immunohistochemistry in hematopoietic and other tissues can be achieved with inexpensive and reproducible steam HIER and that use of mercury-containing B-5 can be discontinued to increase the safety of the laboratory and decrease negative environmental impact. (The J Histotechnol 20:303, 1997)

Image analysis of low magnification images of fine needle aspirates of the breast produces useful discrimination between benign and malignant cases.

Fine needle aspirates of the breast (FNAB) (n = 362; 204 malignant, 158 benign), prepared by cytocentrifuge methods and stained by the Papanicolaou technique, were analysed using a semi-automated image analysis system at a low magnification which precluded resolution of nuclear detail. The measured parameters were integrated optical density, fractal textural dimension, number of cellular objects (single cells and contiguous groups of cells), distance between cellular objects (mean, s.d., skewness and kurtosis), area of cellular objects (mean, s.d., skewness, kurtosis) and the nearest neighbour statistic. The cases were divided into a 200-case training set and a 162-case test set. Analysis was performed by logistic regression and the multi-layer Perceptron type of artificial neural network. Logistic regression and the neural network produced similar performances with a sensitivity of 82-83%, specificity 85% and a positive predictive value for a malignant result of 85%. A non-parametric analysis of all the predictor variables showed that all except the mean area of cellular objects and the s.d. of this measurement were significant discriminants (P < 0.05), but most were highly interrelated and this was reflected in the selection of only three predictor variables by forward and backward conditional logistic regression. This study shows that much diagnostic information is present in low power views of FNAB, and that image analysis could form the basis of a semi-automated decision-support aid.

Transitional (urothelial) cell metaplasia of the uterine cervix: morphological assessment of 31 cases.

Thirty-one cases of transitional cell metaplasia (TCM) of the uterine cervix were studied. The mean age of patients was 62 years (range 30-87); 25 (81%) were postmenopausal and six premenopausal. In 24 of 25 postmenopausal patients, TCM was an incidental finding at operation for a variety of gynecological lesions, and in such patients the distribution of TCM included the endocervical canal, transformation zone, native ectocervix and vagina. In five of six premenopausal patients, TCM was identified in the cervices of women investigated for abnormal Papanicolaou smears. In all six premenopausal patients, as well as in the remaining postmenopausal patient who also presented with an abnormal Papanicolaou smear, TCM was confined to the endocervical canal and transformation zone. TCM was typified by a multilayer epithelium, clearly distinguishable from the atrophic ectocervical epithelium of most postmenopausal patients by its thickness (averaging 12 cells thick). TCM was identified on the surface epithelium or in sequestered endocervical glands resembling Walthard cell nests of the fallopian tube serosa. There was loss of the usual basal "picket fence" layer seen in cervical squamous epithelia; the cells had crowded nuclei oriented vertically and swirling to give a superficial appearance of disorder. Although lack of "differentiation" from the basal to superficial layers was characteristic, in many examples there was an obvious surface umbrella layer. Intraepithelial glandular differentiation and lesions resembling cystitis glandularis of the urinary bladder were occasionally noted on the ectocervix and vagina. Cellular detail included large pale oval nuclei, oriented vertically, with finely stippled chromatin, small inconspicuous nucleoli, and frequently a deep longitudinal groove. Although the nuclear/cytoplasmic ratio was high, cell-to-cell variation was negligible, and mitoses were rare and never atypical, which, with the nuclear details, distinguished TCM from high-grade squamous dysplasias. The nature of the metaplasia and its biological function are unclear. In postmenopausal patients, it presumably arises occasionally as a consequence of the altered hormonal environment of the cervix and vagina and is of interest only for the possibility of misinterpreting it as a high-grade squamous intraepithelial lesion. In premenopausal patients, it may represent a cellular variation of either physiologic or atypical metaplastic squamous epithelium of the cervical transformation zone.

Comparison of ThinPrep and conventional preparations: Nongynecologic cytology evaluation

ThinPrep processing, an automated cytopreparatory method, has been reported to show good correlation with conventional preparations and to reduce the rate of false-negative diagnoses. In a retrospective review of 230 consecutive nongynecologic cytology cases, we compare the ThinPrep (TP) method with conventional preparations (CP). There were 129 fine-needle aspiration (FNA) specimens from various sites, including 51 breasts, 40 thyroids, 14 lungs, 8 livers, and 16 miscellaneous sites. The sources of 101 body cavity fluids included 68 pleural peritoneal effusions, 25 peritoneal pelvic washings, and 8 miscellaneous sites. Each case was evaluated for cellularity, morphologic details, and obscuring background material. Diagnoses of the TP slides were classified as insufficient, normal, benign, suspicious, or malignant. Each case was then correlated with the tissue diagnosis when available. In TP slides, cellular arrangements, nuclear details, and nuclear cytoplasmic ratio were preserved, while blood and diathesis were eliminated. There was no statistically significant difference between TP and CP in the diagnostic categories. However, in six cases of “insufficient for diagnosis” on FNA by CP. TP yielded sufficient cells and tissue fragments for diagnosis. One case each of FNA and body fluid with a diagnosis of “suspicious for malignancy” by CP was considered “positive” on TP slides. The overall sensitivity of TP was 97.6%, and the specificity was 92.9%. The positive predictive value was 93.0%. We conclude that the ThinPrep method shows good correlation with conventional preparations in both FNA and body fluids. Diagn. Cytopathol. 16:368–371, 1997. © 1997 Wiley-Liss, Inc.

Combined use of the "scratch and smear" sampling technique and Ultrafast Papanicolaou stain for intraoperative cytology.

To assess the applicability of Ultrafast Papanicolaou stain (UFP), a 90-second, high-resolution stain incorporating the air-dried rehydration technique, to intraoperative cytology in surgical pathology laboratories. Two hundred sixty-two randomly selected surgical specimens for intraoperative consultations were included in the study. Each specimen was first conventionally prepared by touch imprinting, wet fixation in alcohol and staining by hematoxylin and eosin (H&E); the remaining specimens were then sampled by the "scratch and smear" technique and subjected to the UFP staining protocol. The combined use of the scratch and smear sampling technique and UFP staining was superior to the conventional methods of touch imprinting and H&E staining in every case because of the following features: (1) intact tissue fragments composed of both epithelial and stromal elements rather than epithelial cells only, (2) polychromasia rather than bichromasia, (3) sharper nuclear and cytoplasmic details, (4) histologic criteria applicable to thick tissue fragments due to transparency and flatness and (5) clear background devoid of red blood cells and eosin. The combined use of the "scratch and smear" sampling technique and UFP enhances intraoperative cytology.

Cytologically low grade malignancies: an important interpretative pitfall responsible for false negative diagnoses in fine-needle aspiration of the breast.

Failure to diagnose breast carcinoma by fine-needle aspiration (FNA) is a major obstacle to expanded use of that technique. While the majority of false negative diagnoses are due to inadequate sampling and insufficient specimens, a significant minority of false negative cases result from inaccurate interpretation of adequate material. Low grade carcinomas including lobular, tubular, low grade adenosquamous, and papillary carcinomas appear to account for many of these diagnostic errors. Careful attention to nuclear detail, monomorphism of cell population and the presence of neoplastic cells with retained cytoplasm should allow the recognition of the majority of these neoplasms as malignant by cytologic examination.

Modified Fite Stain for Demonstration of Mycobacterium Species in Tissue Sections

A modified Fite carbon fuchsin stain for Mycobacterium species and other acid-fast bacteria is described. This procedure eliminates uneven stain differentiation and areas of excess stain deposition. Harris hematoxylin is used for the counterstain to allow better nuclear detail. This stain was found to be superior to conventional Fite staining for Mycobacterium species and other acid-fast bacteria that are difficult to visual and for total tissue demonstration. (The J Histotechnol 19:133, 1996)

The effects of mercaptoethanol-formaldehyde on tissue fixation and protein retention.

The study compared the effects of mercaptoethanol-formaldehyde and formaldehyde alone, on tissue fixation and protein retention in human and mouse tissues. Shrinkage of tissues and the penetration rate of the fixatives were assessed. The cross-linking ability of the fixatives was determined by viscometry, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and spectrophotometry, using bovine serum albumin and human haemoglobin. Tissues fixed in buffered 0.0025% mercaptoethanol-4% formaldehyde showed good nuclear and cytoplasmic detail, better than those fixed in buffered 4% formaldehyde. There was no significant difference in shrinkage. A mixture of 0.0025% mercaptoethanol-4% formaldehyde penetrated faster into adult liver than 4% formaldehyde. The mean penetration rate (+/-SE) or coefficient of diffusibility of 0.0025% mercaptoethanol-4% formaldehyde into adult liver was 1.32 +/- 0.01 and that of 4% formaldehyde was 1.12 +/- 0.06 (p < 0.04). Both fixatives diffused more rapidly into mouse liver than into human liver. The cross-linking ability of mercaptoethanol-formaldehyde depends on the concentration of the fixative and the time of fixation. Bovine serum albumin (15%) and 0.1% mercaptoethanol alone formed a gel, whilst electrophoresis showed monomers in the supernatant. Mercaptoethanol (0.1%) also rapidly decreased the absorption at 420 nm, suggesting denaturation. It seems that mercaptoethanol increases the number of thiol groups available to form cross-links with formaldehyde. This study demonstrated that mercaptoethanol-formaldehyde fixed and cross-linked tissues better than formaldehyde at 3 h and 4 h, but not at 1 h and 2 h. The most effective concentration of mercaptoethanol for tissue fixation in 4% formaldehyde is 0.0025%.

Effect of Environmental Concerns, Safety and Cost Containment on Tissue Handling

Abstract Environmental concerns, safety and cost containment have made uS evaluate tissue preservation, processing and recycling. When we randomly reviewed a number of fixatives, Histochoice, Clarke, acetate buffered formalin, zinc formalin and neutral buffered formalin (NBF), we found that all gave good morphological detail on thin sections. However, thicker specimens (over 1 cm) were better preserved in the formalin fixatives. Zinc formalin gave excellent morphological detail but produced coarse nuclear detail for electron microscopy. Acetate buffered formalin was preferable to NBF because it could be neutralized and disposed of down the sink. We also found that the volume of fixative needed for tissue preservation was overestimated, as only enough for complete immersion of the tissue was sufficient. Reducing the number of changes of all solutions on the tissue processor and recycling the containers used in dispensing the formalin fixatives resulted in considerable savings both financially and environme...

Cytology of Gastrointestinal Histoplasmosis

Gastrointestinal (GI) histoplasmosis is a rare manifestation of Histoplasma capsulatum (HC) infection. There are no reports of its cytologic diagnosis in the literature. A search of the cytology and surgical pathology files of the Methodist Hospital uncovered two cases of GI histoplasmosis and histiocytes within cytologic specimens. Papanicolaou-stained endoscopic brushings of an obstructing, apple-core, right colonic mass in a 58-year-old, heterosexual male revealed numerous vacuolated single cells interpreted as suspicious for signet ring cell carcinoma. The resected colon showed granulomatous inflammation with numerous histiocytes containing pale, oval yeasts of HC. The patient was subsequently found to be human immunodeficiency (HIV) positive; this was his first manifestation of the acquired immunodeficiency syndrome. The second patient was a 69-year-old, HIV-negative male with a fungating anal mass suspicious for squamous cell carcinoma. Direct smears showed oval histiocytes with intracellular yeasts of HC. Accurate diagnosis is crucial to patient management and therapy. Careful attention to the nuclear and cytoplasmic details of histiocytes and histiocytelike cells is important to avoid interpretive errors. Diagnostic pitfalls include signet ring cell adenocarcinoma, lymphoma, melanoma, goblet cell carcinoid, malakoplakia and such infections as mycobacteria, Entamoeba histolytica and Calymmatobacterium granulomatis. Ancillary studies, such as microbiologic cultures and immunohistochemical and histochemical staining, can be performed in the appropriate clinical setting.

Nongynecologic cytology utilizing the ThinPrep Processor.

To assess the utility of the ThinPrep Processor (TP) for nongynecologic cytology. We reviewed the number of unsatisfactory specimens from the esophagus, common bile duct, hepatic duct, pancreas, gastric, bronchial wang, vertebra, submandibular area and neck over a one-year period, before and after TP implementation. For a one-year period after TP implementation, the cytologic diagnoses of selected TP specimens with corresponding surgical tissue diagnoses were compared, and the TP slides were reviewed in discrepant cases. The number of unsatisfactory specimens was reduced from 17% to 1% after TP implementations. The cytologic diagnoses of 145 TP specimens were in agreement with surgical tissue diagnoses. However, in 43 cases the cytology and tissue diagnoses were discordant. On review of 26 of the discrepant cases, the majority of TP slides were cellular, with good nuclear and cytoplasmic detail. Discrepancies resulted from sampling errors in 19 cases and TP interpretation errors in 7 cases. In our laboratory, TP is a reliable processor for nongynecologic specimens.

ThinPrep versus conventional smear cytologic preparations in the analysis of thyroid fine-needle aspiration specimens.

Paired fine-needle aspiration specimens were analyzed from 41 surgically resected thyroid nodules, to compare diagnostic accuracy, amount (absent, mild, moderate, or marked) and pattern (diffuse, droplets, or both) of colloid, nuclear detail (poor, satisfactory, or excellent) and cytoplasmic detail (intact or disrupted) in ThinPrep (TP) (Cytyc, Marlborough, MA) versus conventional smear (CS) cytologic preparations. The 41 surgical specimens included 25 colloid nodules, 6 papillary carcinomas, 4 follicular adenomas, 2 minimally invasive (encapsulated) follicular carcinomas, 3 Hashimoto's thyroiditis, and 1 Grave's disease. Both techniques identified seven of the eight carcinomas with the minimally invasive follicular carcinomas categorized as hypercellular follicular nodule, possibly malignant (HCFN). One papillary carcinoma was classified as a HCFN by both TP and CS techniques. The four follicular adenomas were classified as HCFN based on the TP slides. One oxyphilic follicular adenoma, associated with focal lymphocytic thyroiditis, was misinterpreted as Hashimoto's thyroiditis on a conventional smear. Three colloid nodules were interpreted as HCFN based on the TP slides. Two of these were similarly classified based on the conventional smear. ThinPrep slides contained less colloid and the colloid occurred as droplets rather than a diffuse pattern. TP slides had better nuclear detail but more often disrupted cytoplasm. In conclusion, the TP process does alter some cellular features; however, we experienced similar diagnostic accuracy with the TP and conventional smear preparations.

A Comparison of Withdrawn and Nonwithdrawn Brushes in the Diagnosis of Lung Cancer

Brush specimens are one of several methods for establishing a diagnosis of lung cancer at the time of flexible bronchoscopy. Two acceptable methods of retrieving the brush cytology specimen are to withdraw the brush through the working channel of the bronchoscope and to withdraw the brush and bronchoscope as a unit, with the brush protruding through the distal tip of the bronchoscope. In this prospective study 53 patients with proven primary bronchogenic carcinoma underwent bronchoscopy by one senior staff pulmonary physician; there were 32 patients with peripheral tumors and 21 patients with central tumors. The sequence of sampling (withdrawn or nonwithdrawn) was assigned randomly. The specimens were interpreted by one cytopathologist in a blinded fashion and scored for both cellularity (1–4 according to the density of cells and number of quadrants involved) and presence or absence of recognizable malignant cells. Cellular yield was significantly greater for nonwithdrawn brushes [difference = 1.43 ± 1.21 for central tumors (p < 0.001), and difference = 0.65 ± 1.26 for peripheral tumors (p < 0.01)]. Malignant cells were identified in 66.7% (14/21) of withdrawn versus 71.4% (15/21) of nonwithdrawn brushes (p > 0.25; McNemar's test for matched pairs) for the central tumors. Malignant cells were identified in 53.1% (17/32) of withdrawn brushes versus 59.4% (19/32) or nonwithdrawn brushes (p > 0.25) for the peripheral tumors. This lack of a statistically significant difference could be the result of a type 2 error (inadequate sample size to detect a true difference). There were qualitative differences in the cellular preservation and nuclear details in favor of the nonwithdrawn brush specimens. We conclude, however, that withdrawing the brush through the bronchoscope decreases the cellularity of the specimen, but it does not affect the diagnostic accuracy for malignancy in either central or peripheral tumors.

Morphological diversity of Blastocystis hominis in sodium acetate-acetic acid-formalin-preserved stool samples stained with iron hematoxylin.

The objective of this investigation was to study the morphological characteristics of Blastocystis hominis in sodium acetate-acetic acid-Formalin-preserved stool samples. Routinely processed samples were examined for morphological detail, including size, shape, nuclear detail, and central body characteristics. Morphological findings revealing the importance of recognizing B. hominis in the diagnostic laboratory are described.

Rapid Supplementary Fixation in Frozen Sections: Microwave Versus Conventional Fixation: A Double-Blind Comparative Study

The preservation of morphological details in frozen sections subjected to microwave-assisted fixation was compared with that in frozen sections subjected to snap fixation with either formalin/ethanol or ethanol alone. Especially nuclear details were better preserved after short microwave fixation. Ethanol alone gave the poorest results, with loss of nuclear details in the majority of the cases. In this study there was no significant difference between the morphological results obtained with a cheap domestic oven and that with a specialized microwave processor. Microwave-assisted fixation in frozen sections is a simple, rapid method. It is recommended for use in routine laboratories.

The Cytological Features of Invasive Adenocarcinoma of the Cervix Uteri

Seventy cases of invasive adenocarcinoma of the uterine cervix were predicted on cytological grounds and confirmed by histopathology. The cervical smears from these cases were further analysed and classified into well and poorly differentiated adenocarcinoma on the basis of the pattern of the cells in the smear and their nuclear detail. Using this method of analysis it is possible to increase the accuracy of cytology for the diagnosis of adenocarcinoma of the uterine cervix.