Comparison of ThinPrep and TriPath PREP liquid-based preparations in nongynecologic specimens: a pilot study.

ThinPrep (TP) and TriPath PREP (TriP) are two liquid-based cytologic preparations that produce a thin layer of cells. This study compares the diagnostic accuracy and different cytomorphologic alterations produced by these preparations in nongynecologic specimens. Samples from 10 urines (3 urothelial carcinomas and 7 negative), 4 positive serous fluids, and 7 fine-needle aspirates (FNAs) were prepared by both techniques. FNAs represented one each of: Hashimoto's thyroiditis (HT), hyperplastic colloid nodule (HCN), Hodgkin's lymphoma, liposarcoma, chondrosarcoma, squamous-cell carcinoma (SCC) metastatic to the lymph node, and carcinoid tumor. All 5 participants, none of whom had prior knowledge of the clinical history or histologic diagnosis, reviewed and interpreted the slides. Both techniques produced a clean background and were equally accurate in urines, serous fluids, and three FNAs. TriP was slightly more accurate in four FNAs: HCN and HT where colloid and lymphocytes were better represented, SCC where keratin and malignant cells were more readily identified among lymphocytes, and carcinoid which was easier to evaluate on TriP due to less cellular shrinkage and more dispersion of cells between aggregates. TP preparations had more cell shrinkage, and the chromatin was harder to evaluate. Both techniques produced artificial aggregations of lymphocytes, but TriP had a more evenly dispersed single-cell population between aggregates, rendering them easier to evaluate for atypia. TP produced fragmentation of large sheets that were flattened, while TriP contained larger branching sheets in a three-dimensional (3-D) configuration. TP produced a true monolayer of cells that were all spread at the same plane, while in TriP the cells were spread at slightly different planes, requiring frequent focusing of the viewed plane. While both techniques are acceptable for diagnostic purposes, they both introduce new cytomorphologic alterations that pathologists need to recognize. TriP seems superior to TP in FNAs specimens where preservation of architecture and cellular integrity are important considerations.

Salivary gland fine needle aspiration using the ThinPrep technique: diagnostic accuracy, cytologic artifacts and pitfalls.

OBJECTIVE: To retrospectively assess the diagnostic accuracy, cytologic features and pitfalls of ThinPrep (TP) (Cytyc Corporation, Marlborough, Massachusetts, U.S.A.) versus conventional (smear) preparation (CP) in salivary gland fine needle aspiration biopsies (FNABs) and second, to evaluate the reproducibility of the cytomorphologic criteria used in the evaluation of FNABs prepared by CP versus TP. STUDY DESIGN: All salivary gland fine needle aspiration biopsies (SGFNABs) between January 1996 and June 1999 were retrieved from the cytology files of the University of Michigan Hospital. Histologic correlation was identified when available. Two cytopathologists reevaluated the slides for artifacts, cellular preservation, background material, cellularity, and cytoplasmic and nuclear details. RESULTS: Seventy-four of the 134 (55%) cases identified had histologic follow-up. Fifty (68%) cases were processed by TP and 24 (32%) by CP. FNAB processed by TP and CP correctly identified malignancy in 14 and 9 cases, respectively. There were three (4%) false negative cases. These included two acinic cell carcinomas and one mucoepidermoid carcinoma. There were 37 true negative cases (24 TP and 13 CP) and one false positive case of cellular pleomorphic adenoma (cytologic interpretation, mucoepidermoid carcinoma). All discrepant cases were processed using the TP method. The overall specificity and sensitivity were 98% and 88%, respectively. However, specificity and sensitivity for TP-processed SGFNABs were 96% and 82% as compared to a 100% specificity and sensitivity for CP. Additionally, there were 10 (14%) nondiagnostic cases, 8 of which were processed by TP. Cytologic artifacts associated with TP included diminished/distorted extracellular and stromal elements, cellular shrinkage and tissue fragmentation CONCLUSION: The diagnostic accuracy of TP-processed SGFNABs approaches that of the CP. However, there are several artifacts that may lead to erroneous diagnoses. Additional studies, that depend on real-life clinical samples processed by TP are suggested to modify current diagnostic criteria.

Cytologic artifacts and pitfalls of thyroid fine-needle aspiration using ThinPrep: a comparative retrospective review.

BACKGROUND: The ThinPrep Processor has gained popularity as a collection and preparation technique for fine-needle aspiration biopsy (FNAB). Specific cytologic criteria to evaluate ThinPrep preparation (TP) may differ from those of conventional preparation (CP). The authors retrospectively reviewed the quality, cytologic features, and pitfalls of TP versus CP in thyroid FNABs and addressed the cytomorphologic criteria used to evaluate TP specimens. METHODS: Thyroid FNABs received between January 1996-July 1999 were identified from the computer files of the Department of Pathology, University of Michigan (Ann Arbor, MI). Histologic correlation and clinical follow-up were reviewed. The cytology slides were reevaluated for cellularity, cellular preservation, artifacts, background material, architectural integrity, cytoplasmic details, and nuclear details by two observers. RESULTS: Of the 209 thyroid FNABs performed during the study period, TP and CP prepared 127 and 82 cases respectively. Histologic correlation was available in 68 (33%) cases (32 TP and 36 CP). Overall sensitivity was 80% and specificity was 98%. The sensitivity of CP versus TP was 87% and 70%, respectively. Thyroid FNABs prepared by TP, as compared with CP, were characterized by the following: The TP slide 1) allowed assessment of the overall specimen cellularity but not individual passes of an FNAB, 2) contained only "hard" colloid that appeared dense, markedly fragmented, or in droplets, 3) showed crowded, tight, tissue clusters with loss of cellular preservation, especially in the larger aggregates, 4) demonstrated more cell shrinkage, 5) showed increased disruption of the cytoplasm and numerous naked nuclei, 6) occasionally gave nucleoli a more prominent appearance, and 7) was less likely to show nuclear grooves and "pseudoinclusions" in papillary carcinoma. CONCLUSIONS: This study concluded that cytologic features used to evaluate thyroid FNABs prepared by CP may need to be modified when using TP. Awareness of the above-described findings and further studies to evaluate TP are essential to avoid potential diagnostic pitfalls.

Expression of CD44S and CD44v5 is more common in stage III than in stage I serous ovarian carcinomas.

Experimental evidence suggests that attachment of ovarian carcinoma cells to the peritoneal mesothelium involves the interaction between CD44 on ovarian carcinoma cells and hyaluronic acid on mesothelial surfaces. The authors therefore evaluated local and disseminated ovarian serous carcinomas for the expression of standard CD44 and CD44 splice variants CD44v5, CD44v6, and CD44v7/8. The relative amount of hyaluronic acid (HA) in stroma surrounding tumor nests also was studied. Using immunohistochemistry and archival tissue, 14 serous ovarian carcinomas confined to the ovary (stage I) and 14 serous ovarian carcinomas with peritoneal implants and positive peritoneal fluid (stage III) were stained with antibodies to standard CD44, CD44v5, CD44v6, and CD44v7/8. All tissues also were analyzed for HA using a HA binding peptide. Immunostaining was classified as focal or diffuse and graded from 1 to 4 based on intensity. Immunoreactivity for standard CD44 was seen in 5 of 14 (36%) stage I tumors and 10 of 14 (71%) stage III tumors. Similarly, immunoreactivity with CD44v5 was seen in 2 of 14 (14%) stage I tumors and 9 of 14 stage III tumors (64%). Hyaluronic acid was present in the stroma surrounding all stage I and III tumors, but was more intense in the stroma adjacent to metastatic implants from stage III carcinomas. Tumor cells were uniformly negative for intracellular HA. These results suggest that CD44S and CD44v5 are differentially expressed in early (stage I) and advanced (stage III) ovarian serous carcinomas and support previous studies that suggest a role for CD44 and stromal HA in the dissemination of ovarian epithelial cancer.

p53, c-erbB, and Ki-67 expression in ovaries removed prophylactically from women with a family history of ovarian cancer.

Prophylactically removed ovaries from 64 women were compared with those from 30 women with no known family history of ovarian cancer for expression of the p53 tumor suppressor protein, the c-erbB-2 oncoprotein, and for the proliferation antigen Ki-67. All analyses were performed without knowledge of the family history. Ki-67 was expressed in rare nuclei in both the surface and cyst epithelial cells, whereas p53 was expressed in rare nuclei only in cyst epithelial cells. Neither the proportion of positive cases nor the intensity of staining differed between groups. c-erbB2 was not expressed in surface or cyst epithelium in any case from either group. Nuclei of granulosa and granulosa lutein cells expressed both Ki-67 and p53. In conclusion, increased expression of p53, c-erbB2, and Ki-67 was not found in the epithelium of prophylactically removed ovaries, suggesting that increased expression occurs later in the development of carcinoma or invasive tumor evolves too quickly to identify expression of these proteins in preinvasive epithelium.

HER-2/neu oncogene amplification in stage I and stage III ovarian papillary serous carcinoma.

Oncogene amplification has been implicated in the genesis and progression of many cancers. Overexpression of the HER-2/neu proto-oncogene occurs in 20-30% of ovarian epithelial cancers, in which it may be of prognostic significance. Oncogene overexpression is traditionally studied using immunohistochemistry. In this study we used fluorescent in situ hybridization (FISH) to determine HER-2/neu amplification in ovarian papillary serous carcinoma and compared the frequency of amplification in two stages of the disease. Archival tissues from 23 cases of papillary serous ovarian carcinoma (9 cases of stage I and 14 cases of stage III) were analyzed by FISH using a HER-2/neu probe and a chromosome 17 centromere control probe. Determination of the level of amplification was performed according to the standard protocols of the Cytogenetics Laboratory at Rhode Island Hospital. Of the 23 cases successfully analyzed, the frequency of amplification among stage I tumors was 22% (2/9) and the frequency of amplification among stage III tumors was 71% (10/14). These results are significant (P = 0.036). The frequency of stage I tumors among amplified cases was 17% (27/12) and the frequency of stage III tumors among amplified cases was 83% (10/12). This study not only confirms the presence of a subset of ovarian papillary serous carcinoma with HER-2/neu gene amplification, but it also indicates that HER-2/neu oncogene amplification is more likely to be associated with a more advanced stage. Thus, the present data are consistent with the hypothesis that HER-2/neu amplification, similar to HER-2/neu protein over expression, is a prognostic marker of poor outcome.

Trisomy 8 in stage I and stage III ovarian cancer detected by fluorescence in situ hybridization.

Ovarian cancer is the leading cause of death from gynecologic maligancy among women in the United States. In 1997, there were nearly 27,000 ovarian cancer cases with over 14,000 deaths. Recent attempts at early detection of ovarian cancer have been aimed at the identification of biomarkers that would indicate an underlining malignant process or reflect the biological behavior of the tumor. Our previous studies revealed that chromosome 8 copy number abnormality, especially trisomy, is common in several cancers. Archival tissues from 24 cases of papillary serous ovarian carcinoma (10 stage I and 14 stage III) were analyzed by fluorescence in situ hybridization (FISH) with a chromosome 8-specific alpha-satellite probe (Oncor, Gaithersburg, MD). The analysis was done according to standard protocols of the Lifespan Academic Medical Center Cytogenetics Laboratory at Rhode Island Hospital. Twenty-one of 24 cases (87.5%) were found to be trisomic for chromosome 8, if a cutoff point of >/=15% cells with three signals is adopted. Overall, 80% of stage I and 93% of stage III tumors had trisomy 8. This study confirms the presence of a high frequency of trisomy 8 in both early and late stages of the disease and suggests that trisomy 8 may be an early event in the multistep process leading to ovarian cancer. It is of interest to note that a higher frequency of trisomy 8 was found in a higher stage of disease, consistent with our previous results on breast cancer. Thus, additional FISH studies of ovarian tumors for chromosome 8 copy number assessment may be warranted.

Altered surface and cyst epithelium of ovaries removed prophylactically from women with a family history of ovarian cancer.

Despite intensive investigation, the nature of epithelial ovarian cancer precursors remains controversial. Because women with a strong family history of ovarian cancer have a high probability of developing ovarian cancer themselves, ovaries removed prophylactically from such patients provide an opportunity to identify early neoplastic changes. Ovaries removed from 64 consecutive patients undergoing prophylactic oophorectomy and from 30 women with normal ovaries and no known family history of ovarian cancer were examined by light microscopy for a number of histopathologic features and by image cytometry for abnormalities of the cyst and surface epithelium. All analyses were performed without knowledge of the family history. Seven benign, but no tumors of low malignant potential or malignant epithelial tumors were found in the prophylactic oophorectomy group. There were more cortical inclusion cysts in the prophylactically removed than controls ovaries (P = .016), but no other architectural features differed between the two groups. No abnormalities were found in the surface or cyst epithelium in either group by light microscopy. In contrast, image analysis identified differences in the nuclei between the two groups, indicating that those from the surface epithelium of prophylactically removed ovaries were larger and contained more heterogeneously dense chromatin than those of controls, and that nuclei of the cyst epithelium had more irregular outlines. Ovarian epithelium from prophylactically removed ovaries exhibit abnormalities that are only identified by image analysis, and which might represent early preneoplastic changes. Such ovaries may be useful for identifying early molecular changes in ovarian cancer.

Mother's milk protein profile, a possible biomarker for human exposure to persistent insecticides.

The electrophoretic protein profile and residue levels of selected persistent insecticides were investigated in 160 mother's milk samples representing 20 different locations in Egypt. Nine major protein bands were detected in all of the samples. These protein bands were designated as lactoferrin, albumin, SIgA heavy chain, casein I, casein II, SIgA light chain, casein III, lysozyme and alpha-lactalbumin. Residue levels of DDT and its metabolites as well as lindane and its other hexachlorocyclohexane isomers were determined using electron capture gas chromatography and confirmed by gas chromatography/mass spectrometric analysis. Samples containing relatively higher residue levels of the DDT group (DDT, DDE and DDD) showed significant effects on the levels of lysozyme and alpha-lactalbumin bands relative to samples with low or no residue levels. On the other hand, the casein subunits were mostly affected by the residue levels of hexachlorocyclohexane isomers (alpha, beta, gamma and delta isomers). The two patterns showed characteristic dose response correlation suggesting that the protein profile of human milk may serve as a quick biomarker for exposure to persistent insecticides.

Purification and characterization of human serum hyaluronidase.

Hyaluronidase from fresh human serum was purified to apparent homogeneity in a two-step procedure. Potent serum inhibitors of hyaluronidase activity were removed during the course of the purification. Isolation of the enzyme was expedited by the use of a newly devised ELISA-like assay. Enzyme activity was measured by following the rates of hydrolysis of hyaluronan (HA) adsorbed onto microtiter wells. Following enzymatic digestion, the remaining HA was measured using a cartilage-derived biotinylated HA-binding protein and an avidin-peroxidase reaction. Molecular sieve chromatography yielded a doublet of proteins with apparent molecular sizes of 42 and 50 kDa. The molecular size of the major band of protein obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions was 59 kDa. Under reducing conditions, however, the size increased to 72 kDa. The pH optimum of the enzyme was 3.7. Sodium chloride concentrations greater than 100 mM were inhibitory. Activity of the serum enzyme was further characterized with a new HA-substrate gel procedure. The serum enzyme activity is different from the liver-derived activity. The tissue source of this circulating enzyme is unknown.