The use of fine-needle aspiration biopsy in children.

Fine-needle aspiration biopsy has become increasingly popular for evaluating both palpable and nonpalpable masses. Judging from the current body of literature, this procedure is underused by American pediatricians. We report a series of 84 pediatric patients--younger than 16 years--who underwent 92 fine-needle aspiration biopsies to evaluate a large variety of masses. The results of 51 (55%) were benign, and 33 (36%) biopsies revealed malignancy. In two cases (2%) the findings were suggestive of malignancy, and in six cases (7%), insufficient material was rendered for diagnosis. Either histologic or clinical follow-up or both were available for 85 procedures. Based on these cases, the sensitivity of 97% and the specificity was 95%, indicating that fine-needle aspiration biopsy is a good method for screening and observing patients for various malignant tumors in a variety of organs.     

Percutaneous fine-needle aspiration biopsy of intra-abdominal masses.

Percutaneous fine-needle aspiration biopsies were performed in 51 patients with various intra-abdominal masses localized by palpation, radiologic studies, ultrasonography or radioisotope scanning. Biopsy specimens were considered positive for malignant disease in 35 (85%) of the 41 patients with such disease, including 26 (96%) of the 27 with metastases. There was one false-positive diagnosis of malignant disease from the biopsy specimens. Surgery became unnecessary as a result of aspiration biopsy in at least 12 patients. One patient showed evidence of intrahepatic bleeding during liver biopsy but recovered spontaneously, and the liver appeared normal at laparotomy 3 weeks later. Aspiration biopsy is an accurate, relatively painless, inexpensive and safe method of establishing a diagnosis of intraabdominal malignant disease. Considerable experience of the cytologist is necessary for good results.     

Diagnosis of cervical thymoma by fine needle aspiration biopsy with flow cytometry. A case report

BACKGROUND: Cervical thymoma is a rare entity. To our knowledge, this is the 20th reported case of cervical thymoma and the fourth case of fine needle aspiration biopsy (FNAB) of this entity. To our knowledge, this is the only case in which cervical thymoma was a diagnostic consideration at the time of the FNAB diagnosis. The diagnosis was rendered because, unlike in previous cases, flow cytometric immunophenotyping was performed. CASE: A 46-year-old, white female presented with what was clinically thought to be a left thyroid nodule. The patient underwent FNAB at an outside institution, and the diagnosis of "possible mixed lymphoma" was made by morphology alone. The patient was referred to our institution for repeat FNAB. Based upon the cytologic findings (cells with lymphoid morphology), flow cytometry was performed, and a diagnosis of cervical thymoma (versus ectopic thymic tissue) was based upon flow cytometry findings combined with morphology. CONCLUSION: When FNAB of a cervical mass, particularly one clinically thought to be a thyroid nodule, shows lymphoid cells without thyroid follicular cells, immunophenotyping may be extremely helpful in arriving at the correct diagnosis.     

Cytodiagnosis of lymphoid proliferations by fine needle aspiration biopsy. Adjunctive value of flow cytometry

OBJECTIVE: To determine the accuracy of fine needle aspiration biopsy (FNAB) complemented by flow cytometry (FC) for the diagnosis of reactive and neoplastic lymphoid proliferations and subclassification of malignant lymphomas. STUDY DESIGN: Forty-one FNABs of lymphoid lesions on which FC had been performed were evaluated retrospectively. All cases were correlated with histology or clinical follow-up. RESULTS: Twelve FNABs were diagnosed as reactive. Eleven of the 12 were confirmed as reactive on follow-up. One was a case of posttransplant lymphoproliferative disorder. Twenty-five FNABs diagnosed as lymphoma were confirmed by histology. In 22 of these 25 cases, there was 100% correlation between the subclassification given on FNAB with FC and that given on histology. Two of the remaining cases, which were correctly called follicular center cell lymphoma, showed discrepancies in grading. One case called Hodgkin's disease on FNAB was T-cell lymphoma on histology. Of four FNABs given an inconclusive diagnosis, two were lymphoma on follow-up, and two were reactive. CONCLUSION: FNAB examination, when it includes immunophenotyping by FC, is a useful technique for distinguishing reactive lymphoid proliferations from malignant lymphomas and for the subclassification of lymphomas.     

Metaplastic breast carcinoma. A diagnostic problem in fine needle aspiration biopsy.

Fine needle aspiration (FNA) was used to evaluate a breast lump and enlarged lymph nodes in a woman with a prior history of lumpectomy on the contralateral breast and a recent negative mammogram. The FNA cytologic findings included markedly atypical fibroblast-like cells lying singly and in groups in a myxoid background, highly atypical multinucleated cells and numerous mitoses, features that were interpreted as a high-grade malignant mesenchymal tumor. The carcinomatous cells in the aspirates were only fully appreciated after histologic examination of the mastectomy specimen and the axillary lymph node metastases showed a dual differentiation consisting of both epithelial and mesenchymal components, leading to a final diagnosis of metaplastic carcinoma. Electron microscopic study of histologic samples confirmed the dual differentiation, and both keratin and vimentin intermediate filaments were recognized by immunohistochemical staining. The regional lymph node metastases were predominantly sarcomatous, which apparently is a rare event. The entity of metaplastic carcinoma is discussed in relation to other mixed epithelial-mesenchymal lesions of the breast, and the previous literature on this entity is briefly reviewed.     

Secretory carcinoma of the breast. Report of a case with fine needle aspiration biopsy

A case of secretory carcinoma of the breast in a 48-year-old woman studied by fine needle aspiration (FNA) cytology is presented. The prominent intracytoplasmic vacuolization and numerous signet-ring forms and the presence of sheets of neoplastic cells with large intercellular spaces should permit its diagnosis in FNA specimens. The differential diagnosis of this rare lesion from other breast neoplasms that show similar features is discussed.     

DNA distribution in biopsy specimens from human cervical carcinoma investigated by flow cytometry.

Flow cytometry was used for the investigation of the DNA distribution in biopsy specimens from 51 patients with cervical carcinoma. Portio biopsy specimens from 9 pregnant women and from 10 patients with cancer of the breast served as controls. The results demonstrate that most specimens from patients suffering from cervical carcinoma contain considerable cell populations with increased DNA as compared with controls. The possible clinical significance of these findings is discussed.     

DNA flow cytometry of breast carcinoma after acetic-acid fixation

Aqueous acetic acid was used to fix and store specimens of tissue prior to dissociation into nuclear suspensions for flow cytometric quantitation of DNA. The optimum concentration was 20 volumes of glacial acetic acid in 80 volumes of distilled water. Both neoplastic and benign nuclei were easily released from the fixed tissue blocks by slicing and shaking. Residual undissociated tissue was suitable for microscopic examination to confirm its identity. The nuclei fluoresced brightly after staining with propidium iodide, and yielded histograms similar to those from unfixed samples. Acetic-acid fixation resulted in slightly broader G1 and G0 peaks in the DNA histograms in comparison to unfixed cells, but fluorescent debris was less and correlation between the flow cytometric S-phase fraction (SPF) and in vitro thymidine labelling index (TLI) was better than with unfixed cells. Twenty-one of thirty-nine acetic-acid-fixed breast carcinomas had DNA indices in excess of 1.0 (increased nuclear DNA content in comparison to benign cells), and eighteen had DNA indices of 1.0 (normal or near-normal). The SPF was usually in excess of the TLI, but the two were significantly correlated (r = 0.72, P less than 0.0001). However, a significant correlation of SPF with TLI held only for the group with DNA index greater than 1.0. DNA indices greater than 1.0 were associated with high SPF and TLI, and high SPF and TLI each associated with low content of estrogen receptor.     

Fine needle aspiration biopsy of neuroendocrine breast carcinoma metastatic to the thyroid. A case report

BACKGROUND: Tumors showing neuroendocrine differentiation arise in a wide range of organs, and metastatic neuroendocrine tumors may be difficult to differentiate from primary tumors. This report describes an unusual case of metastatic breast carcinoma with neuroendocrine differentiation that presented as a solitary thyroid nodule. The diagnosis was made by fine needle aspiration biopsy (FNAB). CASE: A 52-year-old woman presented with a thyroid nodule and bilateral enlarged supraclavicular fossa lymph nodes. FNAB revealed a neuroendocrine carcinoma. Further questioning revealed that the patient had had a breast carcinoma resected eight years previously. The diagnosis of metastatic neuroendocrine breast carcinoma was established by immunocytochemistry. The patient received antiestrogen therapy but subsequently developed skeletal metastases. CONCLUSION: Neuroendocrine carcinomas from various sites show similar cytologic features. In this case, a diagnosis of breast carcinoma metastatic to the thyroid was suggested by the clinical history and confirmed by FNAB with immunocytochemistry.     

The use of microdialysis to monitor rapid changes in glucose concentration.

As a result of the Diabetes Control and Complications Trial, there is increased emphasis on the importance of blood glucose concentration self-monitoring for people with diabetes. The current methods for this are not ideal, and there are many other possible techniques currently under investigation. One of these techniques is microdialysis, which can be used to analyse subcutaneous interstitial glucose concentrations. A system with high recovery has recently been used to monitor glucose concentrations with sampling over one- or two-hour periods. We have investigated whether this system can be used to monitor rapid changes in blood glucose concentration in healthy volunteers with collection intervals of only ten minutes. The results show that microdialysis can be used to monitor rapidly changing blood glucose concentration, but in some subjects, dialysate glucose lagged behind the whole blood and plasma glucose concentrations to a degree that would be clinically significant. It would therefore be necessary to assess the system, comparing dialysate with plasma glucose concentrations in each individual, prior to use in a clinical setting.     

Utilizing flow cytometry to monitor autophagy in living mammalian cells

Autophagy is a major intracellular catabolic pathway that takes part in diverse biological events including response to amino acid starvation, protein and organelle turnover, development, aging, pathogen infection and cell death. However, experimental methods to monitor this process in mammalian cells are limited due to lack of autophagic markers. Recently, MAP1-LC3 (LC3), a mammalian homologue of the ubiquitin-like (UBL) protein Atg8, was shown to selectively incorporate into autophagosome, thus serving as a unique bona fide marker of autophagosomes in mammals. However, current methods to quantify autophagic activity using LC3 are time-consuming, labor-intensive and require much experience for accurate interpretation. Here we took advantage of the Fluorescence Activated Cell Sorter (FACS) to quantify the turnover of GFP-LC3 as an assay to measure autophagic activity in living mammalian cells. We showed that during induction of autophagy by rapamycin, tunicamycin or starvation to amino acids, fluorescence intensity of GFP-LC3 is reduced in a time-dependent manner. This decrease occurred specifically in wild type LC3, but not in mutant LC3^G120A, and was inhibited by autophagic or lysosomal inhibitors, indicating that this signal is specific to selective autophagy-mediated delivery of LC3 into lysosomes. By utilizing this assay, we tested the minimal nutrient requirement for the autophagic process and determined its induction by deprivation of specific single amino acids. We conclude that this approach can be successfully applied to different cell-lines as a reliable and simple method to quantify autophagic activity in living mammalian cells.     

Utilizing flow cytometry to monitor autophagy in living mammalian cells

Autophagy is a major intracellular catabolic pathway that takes part in diverse biological events including response to amino acid starvation, protein and organelle turnover, development, aging, pathogen infection and cell death. However, experimental methods to monitor this process in mammalian cells are limited due to lack of autophagic markers. Recently, MAP1-LC3 (LC3), a mammalian homologue of the ubiquitin-like (UBL) protein Atg8, was shown to selectively incorporate into autophagosome, thus serving as a unique bona fide marker of autophagosomes in mammals. However, current methods to quantify autophagic activity using LC3 are time-consuming, labor-intensive and require much experience for accurate interpretation. Here we took advantage of the Fluorescence Activated Cell Sorter (FACS) to quantify the turnover of GFP-LC3 as an assay to measure autophagic activity in living mammalian cells. We showed that during induction of autophagy by rapamycin, tunicamycin or starvation to amino acids, fluorescence intensity of GFP-LC3 is reduced in a time-dependent manner. This decrease occurred specifically in wild type LC3, but not in mutant LC3(G120A), and was inhibited by autophagic or lysosomal inhibitors, indicating that this signal is specific to selective autophagy-mediated delivery of LC3 into lysosomes. By utilizing this assay, we tested the minimal nutrient requirement for the autophagic process and determined its induction by deprivation of specific single amino acids. We conclude that this approach can be successfully applied to different cell-lines as a reliable and simple method to quantify autophagic activity in living mammalian cells.     

Short-term cryopreservation of human breast carcinoma cells for flow cytometry

A procedure is described for short-term cryopreservation of primary human tumor cells and tissue slices for later analysis by flow cytometry. Cells were mechanically dispersed into a freezing medium, which was then frozen at either -20 degrees C or -70 degrees C for delayed cell cycle analysis. The results show that a correlation coefficient of greater than 0.95 exists between cell cycle kinetic analyses performed immediately after surgical excision of the tumor and on cells frozen from 1 to 30 days at -70 degrees C in this freezing solution. Somewhat lower levels of correlation exist for cells frozen at -20 degrees C in this freezing medium. This procedure has also been successfully used to preserve freshly isolated breast carcinoma cells shipped from distant laboratories for analysis in the flow cytometer, thus expanding the data base on certain types of breast carcinoma.     

The prognostic value of the aspiration biopsy smear in mammary carcinoma.

The prognostic significance of various morphologic factors (size of nuclei, nucleoli, mitotic frequency, naked carcinoma nuclei, nuclear:cytoplasmic ratio, size of cell clusters, necrotic matter, mucoid matter, and lymphocytes) was studied in aspiration biopsy smears from 494 carcinomas of the breast. A multivariate analysis of clinical and cytologic variables was performed with regard to five-and ten-year survival.     

The use of flow cytometry and cell sorting in a human colon carcinoma model

We investigated the growth characteristics of a human colon carcinoma cell line, WiDr, grown in culture flasks and on chick embryonic skin (CES). WiDr cells labeled in vitro with bromodeoxyuridine (BrdU) and analyzed by combined propidium iodide/Hoechst 33258 fluorescence showed evidence of more BrdU incorporation in early S phase as compared to late S phase. When inoculated on the CES, WiDr cells multiplied and invaded the underlying skin. Morphologic examination showed that with extended culture WiDr cells on the CES undergo progressive structural organization with the development of acini and basal lamina, structures similar to those in in vivo tumors. WiDr cells were labeled with monoclonal antibody to carcinoembryonic antigen (CEA) and the brightest 2% of the population was sorted. When subsequently grown on the CES, the sorted cells formed significantly more acinar structures at 3 and 6 days of culture than an unsorted population grown for a comparable time.     

The exclusive use of flow cytometry to evaluate the antibiotic-susceptibility

Background

Live and injured bacteria cannot be successfully discriminated using flow cytometric methods (FCM) with commercial live/dead staining agents because injured cells have intact cell membranes and are counted as live cells. We previously reported that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro (Microbiol. Immunol. 51:763-775, 2007).

Methods

We report that EMA cleaves the chromosomal DNA of antibiotic-injured, but not live, Listeria monocytogenes. The combination of FCM and EMA treatment was evaluated as a rapid method to discriminate between live and antibiotic-injured L. monocytogenes. Additionally, we evaluated our methodology using blood from pediatric patients infected with other gram-negative and gram-positive bacteria.

Results

For antibiotic-injured, but not live, L. monocytogenes in blood, photoactivated EMA suppressed SYTO9 staining, as the SYTO9 staining of the antibiotic-injured L. monocytogenes was weak compared with that of live cells. Similarly, the rapid and clear discrimination between live and injured bacteria (gram-negative and gram-positive) was performed using the blood of pediatric patients administered antibiotics.

Conclusions

The combination of FCM with EMA treatment is a rapid method for evaluating the susceptibility of live pathogens in infants with bacteremia without the need for bacterial culture.

General significance

This assay is more rapid than other currently available techniques due to the elimination of the time-consuming culture step and could be used in clinical settings to rapidly determine the success of antibiotic treatment in pediatric bacteremia through the discrimination of injured (i.e., susceptible to the administered antibiotics) and live pathogens.     

Acute thyroiditis in a child: misleading result of fine-needle aspiration biopsy

We report a case of acute thyroiditis in a 6-year-old girl, whose initial borderline clinical and sonographic data, coupled with the absence of leucocytes and bacteria on the fine-needle aspiration biopsy, led to the reversal of the initial diagnostic impression of acute thyroiditis and the institution of an inappropriate glucocorticoid treatment. Since both diseases are rare in the paediatric age group and therapy is completely different, we conclude that in borderline cases careful clinical observation and the response to the initial antibiotic therapy should be considered more reliable than any single morphologic or microbiologic result. We also suggest that acute bacterial thyroiditis could be usefully classified into two forms, suppurative and non-suppurative.     

Fine needle aspiration biopsy diagnosis of adenoid cystic carcinoma of the breast. A case report.

A case of adenoid cystic carcinoma arising in the breast is presented. The diagnosis was established on a fine needle aspiration (FNA) biopsy sample with histopathologic corroboration. The aspirate yielded a typical smear composed of round, multilayered clusters of uniform epithelial cells arranged around cores of homogeneous, acellular material. Both the conclusiveness of FNA biopsy in diagnosing this rare mammary neoplasm and its resulting in immediate treatment are stressed.