Fine needle aspiration biopsy of the salivary glands. A five-year experience with emphasis on diagnostic pitfalls.

OBJECTIVE: To review the fine needle aspiration (FNA) findings in 151 patients who presented with salivary gland (both major and minor) enlargement from January 1991 to December 1995 in order to determine its sensitivity and specificity and to study the various pitfalls. STUDY DESIGN: The study group consisted of 77 males and 74 females, 16-98 years old (average 55). One hundred twenty-five aspirates (83%) were from the parotid gland, 23 (15%) from the submandibular gland and 3 (2%) from the soft palate. One hundred thirty-seven cases (91%) were adequate for diagnosis. There were 89 (59%) aspirations done by cytopathologists, 100% of which were diagnostic, and 62 (41%) done by clinicians, 48 (77%) of which were diagnostic. Sixty-eight (45%) cases had histologic confirmation. There were 104 (75.9%) benign, 20 (14.6%) malignant and 13 (9.5%) atypical cytologic diagnoses. RESULTS: Using histology as the "gold standard," the sensitivity of FNA cytology was 91%, with a specificity of 96%. A number of problems were encountered in interpreting some cases, not only in differentiating benign from malignant ones but also in the specific classification of these neoplasms. Problems encountered involved differentiating hematopoietic from non-hematopoietic lesions and interpretation of spindle cell neoplasms, acinic cell carcinoma, mucoepidermoid carcinoma, adenoid cystic carcinoma, lymphoproliferative disorders, postirradiation changes, sialadenitis and atypia in pleomorphic adenoma. CONCLUSION: FNA biopsy of the salivary gland is a sensitive and specific diagnostic tool at our institution. Particular attention to subtle morphologic changes may aid in avoiding pitfalls and arriving at the right diagnosis.

Benign pairs. A useful discriminating feature in fine needle aspirates of the breast.

OBJECTIVE: To determine the diagnostic utility of the finding of naked, bipolar nuclei in fine needle aspirates of breast lesions. STUDY DESIGN: Aspirate smears from 150 cases of histologically proven benign (77) and malignant (73) breast lesions were evaluated for the presence of stripped, bipolar nuclei in the smear background as singlets as well as closely associated pairs. RESULTS: The presence of such pairs was a more specific indicator of benign entities (present in 70% of benign lesions vs. 1% of carcinomas) when compared to single, bare nuclei alone (present in 94% of benign lesions vs. 45% of carcinomas). Large numbers of such "benign pairs" also strongly favored the diagnosis of fibroadenoma within the benign subgroup. CONCLUSION: The presence of bipolar nuclear pairs is a valuable addition to the finding of singlets in the diagnosis of benign breast lesions and their subclassification.

Cytologic grading of fine needle aspirates of breast carcinoma by private practice pathologists.

OBJECTIVE: To determine the reproducibility of two clinically proven grading systems for breast carcinoma assessed by private practice pathologists. STUDY DESIGN: Twenty fine needle aspiration (FNA) slides of histologically proven breast carcinoma were submitted to 15 private practice pathologists practicing in 11 separate groups who interpret cytology as part of their daily practice. The pathologists received the same set of slides. They graded the FNAs using the modified Black (MB) (grades 1-3) and simplified Black (SB) (low grade, high grade) grading systems. Specified criteria and guidelines as well as case samples were provided. RESULTS: There was complete agreement among the 15 pathologists on only one case using the MB grading system as compared to five using the SB grading system. In MB, > or = 10 pathologists were in agreement on 14 cases as compared to 19 cases using SB. There were three cases in MB where the grades ranged from 1 to 3. Also noted in MB was the high number of cases graded 2 (intermediate grade). The predominant comment made by the pathologists was the easier, more objective and practical application of the SB. CONCLUSION: High reproducibility in the cytologic grading of FNA of breast carcinoma can be more readily attained among private practice pathologists using the two-tier SB grading system.

Role of cytology in the intraoperative diagnosis of HIV-positive patients undergoing stereotactic brain biopsy.

OBJECTIVE: To determine the accuracy of cytology in the intraoperative diagnosis of central nervous system (CNS) lesions in human immunodeficiency (HIV)-positive patients. STUDY DESIGN: We prospectively studied 75 cases of computed tomography- or magnetic resonance imaging-guided brain biopsies performed with stereotactic instrumentation and a Nashold biopsy cannula over the course of five years. Biopsy samples were sent for both frozen section and immediate cytologic evaluation. Crush preparations (Papanicolaou and Diff-Quik stained) were used for cytologic assessment. There were 23 cases of progressive multifocal leukoencephalopathy (PML), 8 of toxoplasmosis (toxo), 3 of herpes simplex virus, 26 of lymphoma, 3 of HIV encephalitis, 1 of melanoma, 1 of hamartoma and 10 of nonspecific changes (paraffin section). RESULTS: Using permanent hematoxylin and eosin-stained histologic sections as the "gold standard," frozen section had a sensitivity of 78% and a specificity of 90%, while cytology had a sensitivity of 88% and specificity of 90%. Most of the false negative cases in cytology and frozen section were due to the predominance of necrosis and/or gliosis, present in six cases of toxo. Two of the false positive cases in frozen section (diagnosed as lymphoma) showed toxo, while two false positive cases in cytology (diagnosed as PML) showed only gliosis with negative immunoperoxidase staining for PML in the permanent sections. CONCLUSION: First, cytology had higher sensitivity that frozen section. Second, cytology provided faster results in most instances, primarily due to the nature of specimen preparation. Third, most misdiagnoses occurred in infectious diseases, especially toxo; this should therefore be kept in mind when nonspecific changes with an atypical lymphocytic infiltrate are seen. Fourth, cytology can be an alternative to frozen section for the intraoperative diagnosis of CNS lesions in HIV-positive patients. Another advantage of cytology is elimination of the need for cutting potentially infectious fresh tissue.

Diagnostic significance of signet ring cells in fine-needle aspirates of the breast.

Fine-needle aspiration (FNA) is a reliable and cost-effective procedure in the evaluation and management of breast lesions. One diagnostic dilemma that may sometimes arise is the finding of signet ring cells. The isolated finding of such cells in aspirate smears may be particularly problematic in cases of low cellularity or those with otherwise benign features. Although it is generally held that such cells are almost exclusively associated with carcinoma (particularly the lobular subtype), their significance in FNA smears has never been systematically evaluated. To establish their diagnostic utility, we evaluated aspirate smears from 150 cases of histologically proven benign (77) and malignant (73) breast lesions for the presence of signet ring cells, defined as those containing a prominent intracytoplasmic vacuole with nuclear displacement. Signet ring cells were identified in 71% of malignant cases (75% of ductal carcinomas and 71% of lobular carcinomas), mostly as single cells or within small, loosely cohesive tissue fragments. Such cells also present in 6% of histologically proven benign lesions, most commonly within large tissue fragments. Many of these cells were proven to be vacuolated myoepithelial cells, based on histologic correlation and immunostaining results using anti-muscle-specific actin. On the basis of these findings, we conclude that (1) the presence of signet ring cells within small loose tissue fragments or as single cells in FNA smears should prompt close clinical follow-up (including repeat FNA and perhaps surgical biopsy), regardless of smear cellularity, (2) the presence of signet ring cells in cases of adenocarcinoma does not predict a particular tumor subtype, and (3) rare benign breast lesions may contain signet ring cells, particularly within large tissue fragments, and do not, in isolation, warrant surgical biopsy to exclude malignancy.

Cytomorphology of primary CNS lymphoma: review of 23 cases and evidence for the role of EBV.

Primary non-Hodgkin's lymphoma of the central nervous system (PCNSL) has recently increased in incidence, due primarily to an enlarging immunosuppressed patient population. The pathogenetic role of Epstein-Barr virus (EBV) is of interest due to its established role in other lymphoproliferative disorders in immunosuppressed patients. Twenty-three cases of histologically confirmed PCNSL with corresponding cytology were identified, all obtained under stereotactic guidance. Twenty patients were human immunodeficiency virus (HIV) positive, two were HIV negative, and one was of unknown status. Papanicolaou-stained slides were selected from each case and evaluated for the presence of EBV RNA via in situ hybridization (ISH) utilizing a biotinylated probe specific for EBER 1 RNA, and detected by a conventional streptavidin-peroxidase system. The cases included immunoblastic (12), large cell (10), and mixed small and large cell lymphoma (1). The predominant immunophenotype was B-cell (19), although T-cell (2) and biphenotypic (1) cases were also identified. ISH showed nuclear positivity for EBV RNA in 19 of 23 cases (83%). This study confirms the presence of EBV in PCNSL in immunosuppressed patients and implies a potential etiologic role. The ability to demonstrate EBV RNA in cytologic preparations by ISH also raises the possibility of early identification of high-risk patients through detection of EBV-infected lymphocytes in CSF specimens.

Detection of numerical chromosomal abnormalities by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes on archival cytologic samples.

Fluorescence in situ hybridization (FISH) is rapidly emerging as a tool for analyzing numerical and structural chromosomal abnormalities in both liquid and solid tumors. Most studies make use of fresh samples. To determine the feasibility of detecting numerical chromosomal abnormalities (NCA) by FISH using chromosome-specific probes 8, 12, 17, and X (Vysis, Inc., Downers Grove, IL) on archival cytologic preparations, we studied 23 patient samples, one Papanicolaou-and one Diff-Quik-stained slide per case (46 slides), and two additional unstained slides (fresh ascitic fluids) as controls. Included in this study were nine ascitic fluids (four benign and five malignant), four malignant pleural fluids, three benign bladder washes, and seven malignant fine-needle aspirates (FNA) from various sites. The slides ranged from 1-94 days old. After removal of coverslips using xylene, all slides were destained in a series of alcohol and water washes. Pretreatment of slides with pepsin was followed by the in situ hybridization procedure. Two hundred cells per slide were evaluated for distinct separate signals. Results showed the following: 1) all slides were evaluable except for eight (8/46) which had either too few cells or enough cells but with faint signals, 2) the oldest sample showed distinct signals, 3) previously Diff-Quik-stained slides showed relatively better signals than Papanicolaou-stained slides, 4) samples less than a month old showed relatively better signals, and 5) malignant samples showed various NCA, but not the benign samples. We conclude that FISH on archival cytologic preparation 1) is feasible, although age of the slide is a factor since better signals were seen in those less than a month old, 2) shows better results in previously Diff-Quik-stained slides, and 3) is a tool that can be used in the retrospective study of various liquid and solid neoplasms.

Cytology, flow cytometry, image analysis, and interphase cytogenetics by fluorescence in situ hybridization in the diagnosis of transitional cell carcinoma in bladder washes: a comparative study.

The diagnosis of transitional cell carcinoma (TCC) in bladder washes is a diagnostic challenge to cytology. This study assessed the role of flow cytometry (FCM), image analysis (IA), and interphase cytogenetics by fluorescence in situ hybridization (FISH) as adjuncts in the cytodiagnosis of TCC in bladder washes. Forty separate samples of bladder washes were prospectively evaluated by conventional cytology (CY), FCM, IA, and FISH, and the results were compared with the subsequent surgical biopsy specimens which revealed 26 TCC (3 GR I, 6 GR II, 17 GR III) and 14 benign lesions. Using histology as the "gold standard" and following the previously published criteria for detection of TCC by CY, FCM, IA, and FISH, the concordance rates between histology and CY, FCM, IA, and FISH were 75, 74, 89, and 83%, respectively. CY, FCM, IA, FISH, and histology were concordant in 54% of the cases. The sensitivity of CY, FCM, IA, and FISH were 61, 72, 91, and 73%, respectively, while the specificity were 100, 80, 83, and 100%, respectively. The combined sensitivity of all the parameters was 96%. Interestingly, the false positive cases by FCM and IA showed cystitis. We conclude that IA has the highest sensitivity in detecting TCC in bladder washes followed by FISH, FCM, and CY, while CY and FISH have the highest specificity. This study indicates that FCM, IA, and FISH are useful adjuncts to cytology in the diagnosis of TCC in bladder washes. The finding of DNA-aneuploidy in cystitis warrants further investigation.

Intraoperative diagnostic techniques for stereotactic brain biopsy: cytology versus frozen-section histopathology.

Stereotactic brain biopsy has gained widespread acceptance as a primary diagnostic tool for the evaluation of intracranial lesions. Intraoperative evaluation of such specimens has included the use of both cytological and frozen section histologic techniques. The current study seeks to compare the diagnostic utility of frozen section histopathology and cytology in the intraoperative evaluation of stereotactic brain biopsies in HIV-seropositive patients. Seventy-five HIV-seropositive patients undergoing stereotactic brain biopsy for the evaluation of intracranial lesions were evaluated; intracranial diseases were predominantly infectious or hematologic malignancies. Comparison of frozen section and cytology as a means of intraoperative evaluation showed cytology to have a greater sensitivity (86 vs. 78%), positive (95 vs. 90%) and negative (50 vs. 39%) predictive values and a greater overall diagnostic efficiency (84 vs. 75%) than frozen section. Thus, cytology is a highly effective tool equaling and in some cases surpassing frozen section in terms of sensitivity, predictive value, and overall accuracy. Cytologic examination may often be used as the sole means of intraoperative diagnosis, obviating the need for the freezing and sectioning of fresh tissue and potentially reducing specimen turn around time as well. In other cases, cytology can be used in conjunction with other methodologies for arriving at both intraoperative and final diagnoses in these often difficult cases.

Interphase cytogenetics as an adjunct in the cytodiagnosis of urinary bladder carcinoma. A comparative study of cytology, flow cytometry and interphase cytogenetics in bladder washes.

To determine the role of interphase cytogenetics as an adjunct in the cytodiagnosis of transitional cell carcinoma (TCC) in bladder washes, 40 separate samples were prospectively evaluated by conventional cytology, flow cytometry (FCM) and interphase cytogenetics using fluorescence in situ hybridization (FISH) to detect numerical chromosomal abnormalities involving chromosomes 8 and 12. The cases, all of which had subsequent histologic confirmation, were composed of 26 transitional cell carcinomas and 14 benign lesions. Cytology, FCM and FISH were concordant in 19 of 31 (61%) instances. The false-negative rates of the three parameters were as follows: cytology, 38.5%; FCM, 28.5%; FISH, 27%. The false-positive rates were 0%, 20% and 0%, respectively. The relatively lower sensitivity of cytology is attributed to sampling problems since 7 of the 10 false-negative cases were histologically high grade (grades 2 and 3) and should have been detected easily by conventional cytology if tumor cells were present. Thus, the sensitivity and specificity of FISH in detecting malignant cells of TCC in bladder washes is comparable to that of conventional cytology and FCM. When, as in the present study, there are insufficient numbers of cells for FCM analysis, the detection of numerical chromosomal abnormalities by FISH studies may serve as an adjunct in the diagnosis of TCC in bladder washes.

Simplified nuclear grading of fine-needle aspirates of breast carcinoma: concordance with corresponding histologic nuclear grading and flow cytometric data.

Although histologic grading of breast carcinoma is widely practiced by most pathologists, cytologic grading of fine-needle aspirates (FNA) of this neoplasm is not commonly done. This study addresses the issue of the accuracy of a new classification system, a simplified Nuclear Grading (NG) system based on the criteria proposed by Black et al. (Surg Gynecol Obstet 1955;100:543) in FNA of breast carcinoma. We reviewed 100 cases of breast carcinoma, initially diagnosed by fine-needle aspiration biopsy (FNAB) with subsequent histologic confirmation, consisting of 94 ductal, five lobular, and one medullary carcinoma. NG of Papanicolaou's stained materials were reviewed twice independently by two pathologists and then were compared to the original histologic NG. The concordance rate with histology ranged from 80-90%. Intraobserver reproducibility was 86 and 88%, while interobserver reproducibility ranged from 84-88%. Of the 88 cases with corresponding flow cytometic (FCM) data, there were 35 diploid and 53 aneuploid cases. Fifty-nine (95%) of histologic high NG were aneuploid or diploid with high S-phase fraction (SPF), while 20 (77%) of histologic low NG were diploid with low SPF. This study confirms that nuclear grading of FNA of breast carcinoma using a simplified NG system has a high concordance with histology, has high intraobserver and interobserver reproducibility, and that this grading system correlates well with FCM analysis when tumors are simply divided based on NG as high or low grade.

Comparative study of interphase cytogenetics, flow cytometric analysis, and nuclear grade of fine-needle aspirates of breast carcinoma.

The correlation between DNA ploidy and S-phase fraction (SPF) by flow cytometry (FCM) and the detection of numerical chromosomal abnormalities (NCA) by interphase cytogenetics (IC) involving chromosomes 8 and 12 was studied in 20 human breast lesions (17 breast carcinomas, 2 fibroadenomas, and 1 sclerosing adenosis). Initial diagnosis was performed on fine-needle aspiration biopsy (FNAB) material with subsequent histologic confirmation. FCM was performed on formalin-fixed paraffin-embedded tissue while IC by fluorescence in situ hybridization (FISH) was done on alcohol fixed FNAB materials. Sixteen (80%) cases showed concordance between FCM and IC with respect to the presence or absence of aneuploidy. The remainder of the cases (20%), which were all malignant neoplasms, showed discrepancies between the two methods, all four were DNA-diploid with low SPF by FCM but showed various NCA by IC. Nuclear grades (NG) of all the malignant samples were also evaluated and correlated with both FCM and IC studies. Although a good correlation was observed between NG and FCM, a better correlation was seen between NG and IC. This study shows that although IC by FISH correlated well with FCM analysis, it can detect NCA in DNA-diploid, low SPF tumors. It also correlates well with the NG of the tumor. The increased sensitivity provided by IC in detecting aneuploidy may be of great prognostic significance in low stage, DNA-diploid, low SPF breast carcinomas.

Detection of numerical chromosomal abnormalities in malignant cells in fine needle aspirates by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes.

The feasibility of detecting numerical chromosomal abnormalities (NCA) in malignant cells on fine needle aspirates (FNA) using the fluorescent in situ hybridization (FISH) technique was tested on clinical specimens from patients with various neoplasms. Directly labeled DNA probes specific for chromosomes 8 and 12 were used for in situ hybridization to interphase cell nuclei. Thirty-nine of 42 samples from various sites were evaluable. Based initially on the Papanicolaou-stained slides, there were 32 malignant and 7 benign samples. Blind analysis (200 cells per sample) showed that all benign samples had a normal number of chromosomes, while 27 of 32 malignant samples showed different NCA composed of 5-95% of the cell population and ranging from 1 to 10 chromosome signals per cell. We conclude that interphase cytogenetic cell analysis of FNA by FISH is (1) feasible and gives superior signals for detection of NCA, and (2) relatively simple, with a turnaround time of less than 24 hours. This method may have diagnostic and prognostic application in the study of the biologic behavior of malignant neoplasms.

Monoclonal antibody 44-3A6 as an adjunct in cytodiagnosis of adenocarcinomas in body fluids.

Monoclonal antibody (MCA) 44-3A6 detects a cell-surface transmembrane phosphoprotein frequently expressed by pulmonary adenocarcinoma (AC) and associated with glandular differentiation. This antibody has been found to have utility in assessing routine formalin fixed paraffin-embedded pulmonary neoplasms, as well as the cytopathological evaluation of sputum and bronchial brushings. Recently, it has been shown to be useful in cytological diagnosis of pleural effusions. This study is directed at evaluating its effectiveness in detecting immunoreactive neoplastic cells in body fluids (BF) arising in other tissues. A retrospective cohort of 57 cases was studied, consisting of 36 pleural, 19 ascitic, and 2 pericardial BF. After evaluation of Papanicolaou-stained slides, the BF specimens were immunostained with MCA 44-3A6 using the avidin-biotin-peroxidase complex (ABC) method. In 29 cases, tissue sections of the primary tumors, were also available for immunostaining with MCA 44-3A6. Results showed that 39/42 (93%) of AC BF cases were positive and 28/42 (66%) stained intensely (3-4+) with 75-100% of the AC cells staining in each case. All of the 18 benign and non-AC malignant BF were negative. The staining patterns in the tissue sections of the 29 cases that had corresponding BF samples were similar. We conclude from this study that the MCA 44-3A6 (1) is useful in detecting cells consistent with AC in BF; (2) does not stain inflammatory cells or reactive mesothelial cells, thus helping distinguish reactive from malignant BF; and (3) frequency and pattern of expression in BF parallels its expression in tissue specimens. This study confirms that this MCA is a useful adjunct tool in the cytopathological evaluation of BF.

Detection of numerical chromosomal abnormalities in malignant cells on body fluids by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes.

To detect numerical chromosomal abnormalities (NCA) in malignant cells on body fluids, Fluorescence in situ hybridization (FISH) technique was tested in clinical specimens from patients with metastatic disease. Directly labeled DNA probes specific for chromosomes 8, 12, X, and Y (Imagenetics, Naperville, IL) were used for in situ hybridization to interphase cell nuclei. Fifteen body fluids (BF) from various sites were studied. Based initially on the Papanicolaou-stained slides, there were seven malignant and eight benign samples. Blind analysis (200 cells/sample) showed that all benign samples had a normal number of chromosomes, whereas six of seven malignant samples showed different NCA comprising 5-60% of the cell population ranging from three to 10 chromosome signals per cell. We conclude that interphase cytogenetic cell analysis of BF by FISH is: (1) feasible and gives superior signals for detection of NCA, (2) helpful in detecting malignant cells, (3) relatively simple with a turnaround time of less than 24 hr. This method may have diagnostic and prognostic application in the study of the biologic behavior of malignant neoplasms.