Detection of the HTLV-I gene on cytologic smear slides

OBJECTIVE: To apply the polymerase chain reaction (PCR) for detection of the HTLV-I gene from cytologic smear slides. STUDY DESIGN: Samples were from seven cases of serum anti-ATL antibody (ATLA)-positive T-cell lymphoma and three from ATLA-negative T-cell lymphoma. Six of the seven ATLA-positive cases were confirmed to be ATLL by Southern blotting. From the seventh case a fresh sample for blotting could not obtained. DNA was extracted from the cytologic smear slides of all 10 cases; they had been stained with Papanicolaou or May-Giemsa stain, digested with proteinase K and precipitated with phenol and ethanol. The target sequence in the pX region of the HTLV-I gene was amplified by PCR. RESULTS: All seven ATLA-positive cases, including one that had not yet been confirmed by Southern blotting, showed a single band, as predicted, while the three ATLA-negative cases showed no band. CONCLUSION: If cytologic smear slides are available but a fresh sample is not, the PCR method should provide evidence that the virus is present since in our study sufficient DNA templates were successfully extracted from the stained cytologic smear slides for detection of the virus.     

Application of liquid-based preparation to fine needle aspiration cytology in breast cancer

OBJECTIVE: To examine the performance of liquid-based cytology (LBC) in breast cytology to confirm the diagnosis of carcinoma. STUDY DESIGN: Using cell clusters directly scratched from surgically removed tumor masses, we examined the immunocytochemistry, molecular biology and cytomorphology of the specimens. RESULTS: LBC was very useful for gene analysis and evaluating the immunocytochemistry. The cytologic features of LBC were slightly different from those ofa conventional aspiration cytology smear. CONCLUSION: LBC is a promising method for improving the standardization ofpreparations in breast cytology, although care should be taken to account for its characteristic cytologic features. The quantitative analysis of HER-2 mRNA correlated with the results of immunohistochemistry.     

Potential sampling error in fine needle aspiration biopsy of dedifferentiated chondrosarcoma: a report of 4 cases

BACKGROUND: Dedifferentiated chondrosarcoma is a rare, poorly understood and often fatal sarcoma that usually manifests as a high grade, non-cartilage-producing sarcoma juxtaposed against a low grade chondrosarcoma. A correct diagnosis requires recognition of both components. In the absence of complete resection, rendering a specific diagnosis on small biopsy specimens, such as fine needle aspiration biopsy (FNAB), may be extraordinarily difficult. CASES: We retrospectively reviewed 4 cytology samples (3 primary, 1 metastatic) from 3 patients with dedifferentiated chondrosarcoma, initially analyzed by FNAB, emphasizing the potential for sampling error. Two women, aged 78 and 57 years, both of whom had prior histories of carcinoma, presented with lesions involving the right and left femur, respectively. One 27-year-old man with multiple osteochondromatosis developed a dedifferentiated chondrosarcoma of the left pelvis. Two primary cytologic specimens consisted of moderately cellular smears containing a spindled to polygonal, nonspecific, pleomorphic sarcoma unaccompanied by definite matrix material; 1 of these had a concomitant core needle biopsy (CNB), also demonstrating pleomorphic sarcoma. The third primary cytologic specimen revealed low grade chondrosarcoma, but a concomitant CNB showed only a high grade, non-matrix-producing sarcoma. The last patient developed a metastasis to the opposite femur; FNAB revealed a high grade spindle cell sarcoma. In none of the FNAB or CNB specimens were both low and high grade components of dedifferentiated chondrosarcoma recognized. However, the diagnosis was strongly suspected based on the clinical and radiographic findings. CONCLUSION: Due to sampling error, the diagnosis of dedifferentiated chondrosarcoma may be difficult to establish by cytologic examination alone. Clinical and radiographic correlation is essential for an accurate diagnosis.     

DNA polymerase chain reaction using fine needle aspiration biopsy smears to evaluate non-Hodgkin's lymphoma.

OBJECTIVE: To apply polymerase chain reaction (PCR) analysis to the fine needle aspiration biopsy (FNAB) evaluation of lymphoid proliferations. STUDY DESIGN: We analyzed 37 consecutive archived FNAB malignant lymphoma specimens. Immunophenotypic data from the fine needle aspiration biopsy and excisional biopsy material was available for all specimens. PCR to identify monoclonal rearrangements of the immunoglobulin heavy chain gene, T-cell receptor and translocations involving the bcl-1 and bcl-2 genes was performed. RESULTS: Seventy-eight percent of cases were detected by at least one of these assays. Where DNA analysis was performed on excisional biopsy material, 70% of the cases had identical results; no discordant results for the immunoglobulin heavy chain gene or T-cell receptor were found. In 23% of cases, after review of all available data, a discordant result was thought to be a consequence of a false negative result in DNA analysis of excisional biopsy material. CONCLUSION: These findings indicate that PCR analysis of archived FNAB material, when necessary, provides useful information for diagnosis and staging of malignant non-Hodgkin's lymphomas.     

Fine needle aspiration biopsy of Hashimoto's thyroiditis. Sources of diagnostic error.

OBJECTIVE: To determine the accuracy of cytologic interpretation in the diagnosis of Hashimoto's thyroiditis (HT). STUDY DESIGN: At Ottawa Hospital from 1987 to 1994, 1,638 fine needle aspiration biopsies (FNABs) from thyroid were performed. HT was suggested in 184 FNAB samples taken from 157 patients. Of the 184 aspirates diagnosed with HT, 39 had corresponding surgical specimens taken from 31 patients. A retrospective review of these FNABs and surgical pathology slides formed the basis of this study. RESULTS: In 27 (69%) aspirates, HT was diagnosed on both the FNAB and surgical specimens. In 10 of 27 FNABs an associated lesion was not sampled by FNAB. In four of these 10 aspirates some of the cellular features of HT were misinterpreted, and the possibility of an associated neoplasm could not be ruled out. This resulted in four false positive diagnoses. In 12 (31%) FNABs from nine patients, the cytologic diagnosis of HT was not confirmed histologically. These cases included five Hürthle cell adenomas and one case each of follicular adenoma, nodular goiter, macrofollicular adenoma and malignant lymphoma. This resulted in five false negative diagnoses. CONCLUSION: These results support the value of FNAB in the diagnosis of HT. The presence of hyperplastic follicular cells on FNAB samples from HT may mimic a follicular neoplasm and result in a false positive interpretation. Adequate sampling of the thyroid is important, particularly when there is an associated lesion. The diagnosis of lymphocytic thyroiditis should not be made when only a few lymphocytes are present. Finally, pleomorphic Hürthle cells may be present in aspirates from Hürthle cell neoplasms and underdiagnosed as HT, especially when they are associated with a few lymphocytes.     

Fluorescence in situ hybridization for HER-2/neu amplification of breast carcinoma in archival fine needle aspiration biopsy specimens

OBJECTIVE: To assess the usefulness of fluorescence in situ hybridization (FISH) for HER-2/neu amplification of breast carcinoma in archival fine needle aspiration biopsy (FNAB) specimens. STUDY DESIGN: All FISH performed on formalin-fixed, paraffin-embedded surgical specimens during January 2003-August 2003 at the University of California Irvine Medical Center were selected. Prior FNABs were retrieved. One cytologic slide was destained in each case. The results were compared with those obtained on histologic specimens using the paired t test. RESULTS: FISH was performed on 41 surgical specimens of breast carcinoma. Thirteen patients had prior FNABs that were positive for adenocarcinoma. After hybridization on destained fine needle aspiration slides, no cells were found in 2 cases, and the results were not readable in 2 cases. In the remaining 9 cases, the results, expressed as the ratio of copies of the HER-2/neu gene to copies of the chromosome 17 centromere, were 5.10, 1.14, 1.21, 1.12, 0.74, 1.11, 1.21, 9.87 and 2.4. Results on the corresponding histologic specimens were 5.25, 1.05, 1.13, 1.22, 1.13, 1.12, 1.21, 9.35 and 2.61, respectively. No significant difference was found (p = 0.23). CONCLUSION: HER-2/neu amplification status by FISH can be accurately and reliably evaluated in existing archival cytologic slides.     

Fine needle aspiration biopsy of mixed medullary-follicular thyroid carcinoma. A report of two cases

BACKGROUND: Mixed differentiated thyroid carcinomas are rare tumors, difficult to recognize on fine needle aspiration biopsy (FNAB). Most cases are diagnosed only after histologic investigation. CASES: The cases entailed two cytologic samples and a thyroidectomy specimen. Two FNAB thyroidectomy specimens from a 60-year-old man presenting with a solitary thyroid nodule (case 1) were investigated. Both cytologic samples were referred as atypical, with a mixture of features indicating a proliferating follicular lesion but also containing some characteristics of medullary carcinoma. The serum calcitonin level was borderline. Surgery was recommended because of a suspicion of malignancy. The diagnosis of mixed medullary follicular carcinoma was established after a complex histologic investigation. The tumor was encapsulated, with partly microfollicular architecture. Immunohistochemistry was positive for both calcitonin and thyreoglobulin. Electron microscopy from the formol-paraffin block found neurosecretory granules in many cells. The patient was well one year after the operation. One FNAB and thyroidectomy specimen from a 47-year-old woman with long-treated lymphoplasmocellular thyroiditis (case 2) was investigated. The tumor in case 2 was diagnosed on FNAB as medullary carcinoma. Only after histologic and immunohistochemical investigation was mixed differentiation proven. CONCLUSION: Mixed differentiated thyroid tumors are a diagnostic challenge on fine needle aspiration. Irrespective of their rarity, they can be suspected if combined features are present. FNAB recognition of the medullary component in both cases was of crucial importance. Nevertheless, definitive diagnosis remains a histologic problem due to the necessity for topographic information.     

Papillary Hyperplastic Nodule: Pitfall in the Cytopathologic Diagnosis of Papillary Thyroid Carcinoma

ObjectiveTo identify the pitfalls of overdiagnosing papillary formation as papillary thyroid carcinoma (PTC) in thyroid cytology specimens.MethodsPatients with papillary hyperplastic nodules who had preoperative fine-needle aspiration biopsy (FNAB) were selected for this study. All patients had been diagnosed as having either PTC or lesions suggestive of PTC on preoperative FNAB. Pathology reports, surgical reports, and cytopathology slides were reviewed and analyzed for demographic data, nature of surgery, and pathologic features.ResultsSix women and 2 men with a mean age of 49 years (range, 16-79 years) were included. The lesion size ranged from 1.0 to 3.5 cm. Four patients were diagnosed as having PTC and 4 as having lesions suspicious for PTC. FNAB specimens were available for review in 6 cases. Surgical pathology slides were reviewed in all cases. When cytologic material was evaluated for the morphologic features that led to the misdiagnosis of PTC by comparing it with FNAB specimens of classic variant of PTC, the specimens from these patients showed follicular cells arranged in short, nonbranching papillae in a background of watery colloid and macrophages. The follicular cells were round and demonstrated oncocytic change with nuclear enlargement, prominent central nucleoli, nuclear chromatin clearing, and intranuclear grooves.ConclusionsCaution should be exercised rendering the diagnosis of PTC on FNAB samples when a thyroid lesion shows papillary configurations and oncocytic cells and if convincing nuclear features of PTC are not present. Furthermore, some morphologic features on thyroid aspiration can help differentiate these cases from true PTC. (Endocr Pract. 2008;14:863-868)     

Nodular sarcoid myositis of skeletal muscle diagnosed by fine needle aspiration biopsy. A case report

BACKGROUND: Symptomatic striated muscle involvement in sarcoidosis is rare. Muscle biopsy is usually required for the diagnosis. Fine needle aspiration biopsy (FNAB) has been successfully used in diagnosing soft tissue lesions. To the best of our knowledge, FNAB of sarcoid myositis has not been reported. CASE: A 31-year-old, black female with a history of sarcoidosis presented with an enlarging, painful, left calf mass. Infected thrombi were suspected. FNAB showed numerous loosely arranged epithelioid histiocytes, multinucleated giant cells and skeletal muscle cells. The overall cytologic picture was that of granulomatous myositis. The cytologic features coupled with the patient's history and magnetic resonance imaging findings suggested sarcoid myositis. Subsequent muscle biopsy showing noncaseating granulomata and negative stains for organisms confirmed the diagnosis of nodular sarcoid myositis. CONCLUSION: Nodular sarcoid myositis can be suggested by FNAB cytology in a patient with a past history of sarcoidosis.     

Cytologic features of granulosa cell tumors in fluids and fine needle aspiration specimens

OBJECTIVE: To describe the cytologic features of granulosa cell tumors in fluids and fine needle aspiration specimens, with histologic confirmation. STUDY DESIGN: Histologically confirmed granulosa cell tumors, 6 adult type and 1 juvenile type, were identified. All patients had local recurrences or metastases. Eleven specimens from 7 patients, including cytologic samples, cell blocks and histology, were reviewed. Inhibin immunostaining was performed on cell blocks to aid identification of this group of tumors in the cytologic and histologic samples. RESULTS: The patients were 22-72 years old. Sites included ovary and peritoneum; there were pelvic recurrences and metastatic lesions in the spleen, liver, perirectum and cervical lymph node. Cytologic features of adult granulosa cell tumors included 3-dimensional clusters, resettes loose monolayers and individual cells. Other features were Call-Exner bodies, vacuolated cytoplasm, exuberant capillaries associated with papillarylike fronds, a second population of elongated theca cells, and prominent or rare nuclear grooves. In juvenile granulosa cell tumor the features observed were monolayers, loosely cohesive sheets, single cells, occasional larger pleomorphic cells with nuclear clefting and nuclear protrusions, vacuolated cytoplasm, finely granular chromatin and frequent mitoses. The overall cytologic and histologic correlation was good. Inhibin was focally positive in one peritoneal fluid, correlating with the focal pattern of staining seen on histology. CONCLUSION: A definitive cytologic diagnosis of granulosa cell tumor can be made based on the above criteria. Aggressive tumors are discohesive and show pleomorphism and nuclear protrusions. Inhibin stain may be helpful in identifying granulosa cell tumors in cell block specimens.     

Comparison of the pick-and-smear and Saccomanno methods for sputum cytologic analysis

The two methods of preparing sputum specimens for cytologic study, the (fresh) pick-and-smear technique and the (blended) Saccomanno technique, were compared using 249 consecutive specimens. Two slides were prepared for each specimen by each technique. Of the specimens, 103 showed squamous metaplasia, carcinoma in situ or carcinoma. A semiquantitative rating system (0 to 4+) was used to determine the number of diagnostic cells for each method for those 103 cases. More diagnostic cells were found on the Saccomanno preparations (217) than on the fresh preparations (154). There were 121 diagnostic cells in the Saccomanno preparations versus 95 diagnostic cells in the fresh preparations from 63 squamous metaplasias; 7 versus 3 for the preparations from 5 carcinomas in situ; 64 versus 42 from 28 squamous cell carcinomas; 3 versus 1 from 1 large cell undiffernomas; and 12 diagnostic cells in Saccomanno preparations versus 5 in fresh preparations from 3 small cell cancers. Twelve squamous metaplasias, two carcinomas in situ, four squamous carcinomas, one adenocarcinoma and one small cell cancer had no diagnostic cells on the fresh preparations; four squamous metaplasias and one squamous carcinoma had no diagnostic cells on the Saccomanno preparations. More diagnostic information and fewer false-negative results were achieved with the Saccomanno technique.     

Fine needle aspiration biopsy with adjunct immunohistochemistry in intraocular tumor management

OBJECTIVE: To assess the effectiveness of fine needle aspiration biopsy (FNAB), with and without immunohistochemistry (IHC), in the management of solid intraocular tumors. STUDY DESIGN: Thirty-three consecutive adults undergoing FNAB of suspected intraocular tumors were studied. Clinical, cytologic and histologic diagnoses were correlated. The positive predictive value, sensitivity and specificity of FNAB for detecting malignancy, the effect of lHC on the final cytologic diagnosis and the number of patients in whom clinical management was altered as a result of cytologic evaluation were determined. RESULTS: The positive predictive value was 96% with and 93% without adjunct IHC. The sensitivity and specificity of FNAB for detecting malignancy were 96% and 83%, respectively, with IHC. Without IHC, the sensitivity was unaltered, but the specificity was 67%. IHC confirmed the morphologic diagnosis in 75% of cases, made a diagnosis in 12.5% and changed a malignant diagnosis from carcinoma to melanoma in 6% of cases. The planned management was changed by the FNAB findings in 24% of patients. In 3 patients (9%), IHC was essential for diagnosis and management. No patients exhibited local tumor dissemination or recurrence associated with the biopsy. CONCLUSION: FNAB is a safe, sensitive and specific method of establishing a tissue diagnosis in a subset of patients with solid intraocular tumors. The routine use of immunohistochemical stain ing increases the diagnostic utility of the technique and may change clinical management.     

Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children

The broad-range PCR has been successfully developed to search for fastidious, slow-growing or uncultured bacteria, and is mostly used when an empirical antibiotic treatment has already been initiated. The technique generally involves standard PCR targeting the gene coding for 16S ribosomal RNA, and includes a post-PCR visualisation step on agarose gel which is a potential source of cross-over contamination. In addition, interpretation of the presence of amplified products on gels can be difficult. We then developed a new SYBR Green-based, universal real-time PCR assay targeting the gene coding for 16S ribosomal RNA, coupled with sequencing of amplified products. The real-time PCR assay was evaluated on 94 articular fluid samples collected from children hospitalised for suspicion of septic arthritis, as compared to the results obtained with bacterial cultures and conventional broad-range PCR. DNA extraction was performed with the automated MagNa Pure system. We could detect DNA from various bacterial pathogens including fastidious bacteria (Kingella kingae, Streptococcus pneumoniae, Streptococcus pyogenes, Salmonella spp, Staphylococcus aureus) from 23% of cases of septic arthritis giving negative culture results. The real-time technique was easier to interpret and allowed to detect four more cases than conventional PCR. PCR based molecular techniques appear to be essential to perform in case of suspicion of septic arthritis, provided the increase of the diagnosed bacterial etiologies. Real-time PCR technique is a sensitive and reliable technique, which can replace conventional PCR for clinical specimens with negative bacterial culture.     

HER-2/neu evaluation by fluorescence in situ hybridization on destained cytologic smears from primary and metastatic breast cancer

OBJECTIVE: To evaluate HER-2/neu amplification by fluorescence in situ hybridization (FISH) (HER-2/neu by FISH) on archival cytologic smears stained with May-Grünwald-Giemsa (MGG) stain. STUDY DESIGN: Cytologic specimens from 69 breast cancer lesions (48 primary and 21 metastatic), stained with MGG stain for routine diagnostic cytology, were destained and subjected to HER-2/neu by FISH. Fifteen of the 69 samples were also evaluated by FISH on paired fresh smears. RESULTS: HER-2/neu by FISH was successfully assayed in 25 of the 48 primary tumors and in 15 of the 21 metastatic lesions, corresponding to an overall feasibility of 58%. These cases had been archived between 1 month and 10 years prior to FISH analysis. Eight of the 25 primary and 5 of the 15 metastatic tumors were amplified. In 15 of the 40 evaluable cases, HER-2/neu was also assessed on the corresponding fresh smears: 8 tumors were amplified and 7 unamplified on both destained MGG and fresh smears. CONCLUSION: HER-2/neu can be detected by FISH on routinely MGG-stained cytologic slides. This approach allows HER-2/neu evaluation whenever histologic sections or fresh cytologic material are not available. In these cases, HER-2/neu assessment on destained cytologic smears plays a role in the selection of targeted therapy.     

Combined hepatocellular-cholangiocarcinoma. Diagnostic challenge in hepatic fine needle aspiration biopsy

OBJECTIVE: To study the cytohistologic features of combined hepatocellular-cholangiocarcinoma (CHCC-CC) in fine needle aspiration biopsy (FNAB) material. STUDY DESIGN: Six hepatic FNAB cases with cell blocks (five) and hepatic resections (two) were analyzed cytohistologically and immunohistochemically. RESULTS: The six cases were diagnosed as CHCC-CC based on clinicopathologic correlation. Unequivocal hepatocellular carcinoma (HCC) cells corresponding to Edmondson and Steiner's grade 3 lesions were identified in the FNAB in three instances. Adenocarcinoma, represented by cohesive columnar cells with ovoid, basal nuclei displaying nuclear palisading, acini and/or papillary structures with variable intracytoplasmic intraacinar or brush border mucin production, was identified in all cases. Intermediate cells with hybrid/polymorphic cytologic features straddling malignant hepatocytes and glandular cells were identified in five instances. Tissue alpha-fetoprotein was negative. There was brush border and/or diffuse cytoplasmic p-carcinoembryonic antigen immunoreactivity in the glandular elements. CONCLUSION: FNAB diagnosis of CHCC-CC is possible if the clinical, cytohistologic and immunohistochemical findings support the presence of HCC and adenocarcinoma. Intermediate cells pose a great challenge to recognize and define: they tend to lose the classic cytologic features of malignant hepatocytes and acquire glandular characteristics. At the very least, there should be a high index of suspicion. These cases underscore the necessity for clinicopathologic correlation in enhancing the precision of FNAB diagnoses.     

Analysis of KIT mutation and protein expression in fine needle aspirates of gastrointestinal stromal/smooth muscle tumors

OBJECTIVE: To determine if sequencing the KIT gene could facilitate more definitive FNA diagnosis. STUDY DESIGN: Sixteen cases of gastrointestinal stromal/smooth muscle tumor (GIST) in which fine needle aspiration (FNA) was performed (mean age, 67; M/F = 12/4) were studied. DNA was extracted from cytologic preparations from all patients (15 cell blocks, 1 alcohol-fixed smear) and seven subsequent resection specimens. DNA was amplified by polymerase chain reaction, using primers designed to amplify a segment of the KIT gene exon 11 and sequenced on an ABI Prism 377 DNA sequence analyzer (Applied Biosystems, Indianapolis, Indiana, U.S.A.). Immunocytochemical staining for CD 117 (the KIT gene product) was performed on sections from 12 cell blocks and 7 surgical resections. RESULTS: In-frame deletion of exon 11 was detected in eight cases (7 monoalleic, 1 bialleic); a point mutation was found in one case. Mutation was found only in histologically malignant (6 of 10 cases) and borderline GISTs (3 of 4 cases). No mutation was identified in benign tumors. In three cases, scant cellularity or blood precluded sequencing. CD 117 was expressed in 12 of 15 cases. CONCLUSION: Immunocytochemical staining for CD 117 is useful in confirming a cytologic diagnosis of GIST but does not facilitate diagnosis of malignancy. FNA biopsy specimens are suitable for KIT gene sequencing; detection of a KIT mutation favors a malignant diagnosis, though absence of mutation does not preclude malignancy.     

Extraction of DNA from exfoliative cytology specimens and its suitability for analysis by the polymerase chain reaction

The extraction of DNA from archival exfoliative cytology samples would allow the molecular biological analysis of this readily available material using the polymerase chain reaction (PCR). We have quantitatively and qualitatively studied the extraction of DNA from a variety of cytological preparations. For both fresh and archival cervical smears, overnight incubation with proteinase K produces high yields of high molecular weight DNA, but simply boiling the samples produces DNA suitable for PCR amplification of a single copy gene. Increasing the proteinase K incubation to several days allows the extraction of DNA from fixed and stained archival cytology slides from a variety of sites. The extracted DNA was again suitable for PCR analysis. Fresh and archival cytological material can be utilized for molecular biological study of disease processes using PCR. Archival cytological material is probably the best source of DNA and RNA after stored frozen tissue.     

Application of the immunoperoxidase technique to cell block preparations from fine needle aspirates

Immunocytochemistry on fine needle aspiration (FNA) material has been mainly performed on cytologic preparations; there have been few reports on the use of FNA cell blocks. This study compared the intensity scores of immunoperoxidase staining on FNA cell block preparations from 21 breast, 12 thyroid and 10 lymph node aspirates with the scores on the corresponding surgically excised specimens. FNA materials for cell blocks were fixed in formalin and embedded in agar. Ten commercially available antibodies forming three panels were studied using standard peroxidase-antiperoxidase and avidin-biotin complex techniques. In general, the staining results on the FNA cell block sections agreed with those on the surgical specimens; in addition, there were fewer aberrant positive staining results and much less background staining in the cell block sections. These phenomena were most striking with the cytokeratin antibodies. It is concluded that immunoperoxidase staining on FNA cell block preparations is reliable; the advantages of the use of cell block sections as opposed to smears are discussed.