Species-specific immunocytochemical localization of Mycobacterium tuberculosis complex in fine needle aspirates of tuberculous lymphadenitis using antibody to 38 kDa immunodominant protein antigen

OBJECTIVE: To examine immunocytochemical localization of Mycobacterium tuberculosis (MTB) complex antigen in fine needle aspiration (FNA) smears of tuberculous lymphadenitis (TBLN) using species-specific monoclonal antibody MTSS to 38-kDa immnunodominant protein antigen as a diagnostic adjunct to conventional cytomorphology and its advantage over Ziehl-Neelsen (ZN) microscopy. Study Design FNA smears from 340 cases-174 TBLN; 34 negative controls from nontuberculous, positive controls of 13 known acid-fast bacilli (AFB)-positive sputum smears; 50 blind controls; and 69 other controls (smears from stock cultures of bacterial, atypical mycobacteria and fungal species) were subjected to ZN and immunocytochemical staining using MTSS by the streptavidin-biotin method. RESULTS: Immunocytochemical staining was positive in 59 of 61 (96.7%) archival and 110 of 113 (97.3%) fresh FNA smears; ZN positivity for AFB was observed in 27 of 61 (44.2%) archival and 48 of 113 (42.4%) fresh FNA smears of TBLN. CONCLUSION: The immunostaining using MTSS showed a definite advantage over conventional ZN staining for detection and specific diagnosis of TBLN in FNA smears with 0% false positive results. Immunostaining of cytosmears with species specific antibody to MTB would prove to be a good diagnostic adjunct to morphologic diagnosis.     

Dot-ELISA vs. PCR of fine needle aspirates of tuberculous lymphadenitis: a prospective study in India

OBJECTIVE: To evaluate an in-house dot-enzyme-linked immunosorbent assay (ELISA) for confirmation of clinically suspected cases of tuberculous lymphadenitis (TBLN). STUDY DESIGN: The study was performed at the postgraduate departments of microbiology and pathology of a tertiary-care teaching hospital in India. Suspected cases of TBLN were prospectively enrolled. Fine needle aspiration was done of enlarged lymph nodes in all patients, and 2 smears were prepared, 1 for acid-fast bacillus (AFB) demonstration and the other for cytologic examination. The remaining material was tested with in-house dot-ELISA and by IS6110 amplification with polymerase chain reaction (PCR), for diagnosis of TBLN. RESULTS: ELISA was more sensitive and detected 93.2% of cases. PCR and fine needle aspiration cytology (FNAC) detected 82.5% and 61.0% cases, respectively. AFB positivity was 33.1%. CONCLUSION: Application of dot-ELISA was more sensitive but less specific as compared to PCR. PCR, though expensive, should be used in problem cases because of its high specificity.     

Critical appraisal of fine needle aspiration cytology in tuberculous lymphadenitis.

A cytomorphologic diagnosis of tuberculous lymphadenitis by examination of needle aspirates was made in 560 of 1,471 cases of lymphadenopathy studied over two years. Cytologic features were categorized into four groups: epithelioid clusters with or without Langhans's giant cells without necrosis (32.14%), epithelioid clusters with or without Langhans's giant cells with necrosis (50.35%), occasional epithelioid cells without characteristic necrosis/giant cells (2.85%) and necrosis without epithelioid clusters or Langhans's giant cells (14.64%). While a diagnosis of tuberculous lymphadenitis was offered with confidence in the first two groups, constituting about 82.49% cases, aspirates from the third- and fourth-group patients were subjected to Ziehl-Neelsen staining for acid-fast bacilli, which was positive in 12.5% and 75.6% of cases, respectively.     

Polymerase chain reaction vs. conventional diagnosis in fine needle aspirates of tuberculous lymph nodes

OBJECTIVE: To compare four conventional methods of diagnosing tuberculous lymphadenophathy (TL)--namely fine needle aspiration cytology (FNAC), Zeihl-Neelsen staining of smears for acid-fast bacilli (AFB), culture for Mycobacterium tuberculosis (MTB) and lymph node biopsies--with the polymerase chain reaction (PCR) in order to assess the practicability and advantage of its use in routine diagnosis in a developing country. STUDY DESIGN: Fine needle aspirates from 142 consecutive patients presenting with lymphadenopathy (mainly cervical) without any known systemic involvement underwent cytomorphologic diagnosis, AFB smears, culture for MTB, confirmatory biopsy and PCR for MTB. The aspirates from cases other than TL served as controls for PCR. RESULTS: Correct diagnosis of tuberculosis could be made in 94.87% of cases by a combination of the four methods. PCR was done in 52 cases, 39 confirmed TL and 13 controls. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value of PCR were 94.44%, 38.23%, 44.73% and 92.85%, respectively, when culture alone was considered the gold standard. However, specificity (38.23-92.30%) and PPV (44.73-97.36%) of PCR increased remarkably when response to treatment was taken as the final arbiter. CONCLUSION: The four conventional tests were found to be the methods of choice for the diagnosis of TL in developing countries. PCR should be reserved for problem cases.     

Flow cytometric DNA ploidy analysis of soft tissue sarcomas. A comparative study of preoperative fine needle aspirates and postoperative fresh tissues and archival material

Flow cytometric (FCM) DNA ploidy measurements on frozen fresh samples of soft tissue sarcomas were compared with the corresponding analyses on preoperative fine needle aspirates and postoperative formalin-fixed archival tissues from the same tumors. A concordance in ploidy status (diploid versus non-diploid) was obtained for 63% of the fresh tissue-fine needle aspiration (FNA) sample comparisons and for 85% of the fresh tissue-archival material comparisons. The majority of discordances in the fresh tissue-FNA sample comparisons could be explained by FNA sampling errors. In the remaining discordant cases (3 of 27 FNA sample comparisons and 6 of 40 archival material comparisons), sampling errors could not explain the differences in ploidy status. The discordant cases were evenly distributed among the different sampling methods. Method reproducibility was not responsible for the differences in ploidy determinations; tumor heterogeneity may be an explanation for the discrepancies. This study showed that archival soft tissue sarcoma samples are as well suited for DNA ploidy analysis as are fresh frozen tissues.     

Genotypic analysis of DNA isolated from fine needle aspiration biopsies

DNA was isolated from 20 fine needle aspiration (FNA) biopsies from lymphomas, hyperplastic lymph nodes and nonlymphoid malignant tumors. Small aliquots (0.2 microgram to 2.0 micrograms) of DNA from each sample were digested to completion with restriction endonuclease Eco RI and/or Bam HI and electrophoresed in 0.8% agarose minigels. DNA was transferred to a nylon filter after brief treatment in HCl and subsequent denaturation and neutralization. Filters were hybridized to radiolabeled JH, C kappa, TCR beta or bcl-2 probes to determine if these genes were in germline or rearranged configurations in each of the samples. It was possible to demonstrate rearrangement of at least one immunoglobulin gene in each of the samples diagnosed as lymphoma, while all samples derived from hyperplastic lymph nodes and nonlymphoid malignant tumors exhibited a germline pattern for each probe tested. Thus, FNA biopsies can provide suitable and sufficient DNA for genotypic analysis using molecular probes that detect gene rearrangement.     

Polymerase chain reaction for Mycobacterium tuberculosis complex DNA. Use on negative archival Ziehl-Neelsen cytologic samples

OBJECTIVE: To evaluate the usefulness of a nested polymerase chain reaction (PCR) for Mycobacterium tuberculosis complex on routinely stained cytologic samples from patients with extrapulmonary tuberculosis. STUDY DESIGN: Nested PCR for the detection of a fragment of the IS6110 insertion sequence of M tuberculosis complex was applied to Ziehl-Neelsen-negative archival cytologic slides of serous effusions (pleural [n = 7], peritoneal [n = 1] and pericardial [n = 1]) and a lymph node fine needle aspirate (n = 1) from nine human immunodeficiency virus (HIV)-positive patients with autopsy-proven active extrapulmonary tuberculosis. Malignant effusions and aspirates from nine HIV-positive patients with non-Hodgkin's lymphoma and pleural effusions from seven HIV-negative patients with heart failure were used as controls. DNA was extracted after removing the coverslip and gently scraping the cytologic sample from the slides. RESULTS: In all cases, enough DNA was obtained for PCR without any significant loss of integrity, as demonstrated by PCR positive for HLA-Dq. PCR for M tuberculosis was positive in 8 of the 10 samples (80%) from patients with tuberculosis but also in three samples (30%) from HIV-positive patients in the control group. None of the samples from the HIV-negative patients was positive. CONCLUSION: PCR for M tuberculosis can be reliably performed on archival cytologic slides from extrapulmonary samples, but although it is highly sensitive, it may lead to positive results in immunocompromised patients without any sign of active tubercular disease.     

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.     

Acid-fast bacilli in fine needle aspiration smears from tuberculous lymph nodes. Where to look for them

OBJECTIVE: To analyze where acid-fast bacilli (AFB) are most often seen in smears prepared from tuberculous lymph nodes. STUDY DESIGN: Patients referred for fine needle aspiration cytology for evaluation of lymphadenopathy between March 1994 and June 1998 were analyzed. Only those cases clinically and therapeutically proven to be tuberculous were included in the study. RESULTS: Of 783 cases analyzed, 213 (27.2%) were tuberculous. Aspirates obtained were of three types: blood-mixed particles, caseous material and pus. Five cytologic pictures were seen: epithelioid cell granulomas alone or with coexistent necrosis, AFB or both, and necrosis with AFB. AFB were most often seen in purulent aspirates, followed by caseous and least often in blood-mixed particles. Granulomas were most often seen when the aspirate was blood-mixed particles, followed by caseous and, least often, pus. CONCLUSION: AFB detection should be carried out on all suspected tuberculous patients. The relationship between the presence of granuloma and of AFB is inverse. The chance of finding AFB is highest in patients presenting with a cold abscess and yielding pus on aspiration followed by patients who yield caseous material on aspiration.     

Cytomorphology of tuberculous mastitis. A report of nine cases with fine needle aspiration cytology

Nine cases of tuberculosis of the breast were diagnosed by fine needle aspiration (FNA) cytology over a six-month period. All cases presented with a breast lump, three of which clinically simulated carcinoma. FNA cytology showed a picture of suppurating granulomatous mastitis; the diagnosis of tuberculosis was established after the demonstration of acid-fast bacilli in the aspirated material.     

Evaluation of fine needle aspiration vs. fine needle capillary sampling on specimen quality and diagnostic accuracy in endoscopic ultrasound-guided biopsy

OBJECTIVE: To compare percutaneous and endoscopic ultrasound (EUS)-guided biopsy techniques. Study DESIGN: From July 2005 to February 2006, all patients referred for EUS-guided fine needle aspiration (FNA) were considered. If inclusion criteria were met, the first 2 biopsy passes were performed without suction (fine needle capillary [FNC] sampling). Two additional passes were performed using the same needle with 10 mL of applied suction (FNA). A single blinded pathologist later retrospectively evaluated each set of slides. Fifty-three patients met inclusion criteria. The study group comprised pancreatic masses (23), lymph nodes (26), subepithelial masses (3) and liver lesion (1). There were 38 malignant and 15 benign lesions. RESULTS: No statistically significant differences were found with the scoring systems considered in the study. In the subgroups of patients with pancreatic masses, lymph nodes, benign disease and malignant disease, no statistically significant outcomes were noted. CONCLUSIONS: No difference exists between quality and diagnostic accuracy of specimens obtained from EUS-guided tissue acquisition via FNC and FNA. The decision to use FNC or FNA should be left to the discretion of the individual endosonographer.     

Method of extracting DNA from fine needle aspirates of human solid tumors for Southern blot analysis

A rapid, simple, convenient method for extracting DNA from fine needle aspiration (FNA) samples of human solid tumors for Southern blot hybridization studies is described. After the preparation of an air-dried cytologic smear, the remaining sample in the needle was rinsed directly into a test tube for DNA extraction. The extraction procedure, in which manipulation of the sample is minimized, produced sufficient DNA for Southern blot analysis within 24 hours of the FNA biopsy in the ten consecutive cases studied. The DNA bound to the nylon membranes can be washed and reexamined with a variety of probes, allowing studies of lymphoid cell lineage, oncogene amplification or tumor progression. The assessment of cellularity on the cytologic specimen at the time of FNA provided a reliable guide to the need for further passes to obtain sufficient cells for DNA hybridization; the cytologic diagnosis could also be made on the smears.     

Heterogeneity of DNA distribution pattern in colorectal carcinoma. A microspectrophotometric study of fine needle aspirates

For 50 consecutive patients with colorectal carcinoma, the nuclear DNA content in four separate fine needle aspiration samples taken from the resected tumors was analyzed using single-cell Feulgen cytometry. The DNA distribution patterns were divided into four classes according to their degree of euploidy/aneuploidy. Twenty tumors (40%) displayed a similar DNA pattern in all four samples while 30 tumors (60%) were heterogeneous, with a different DNA pattern within one or more of the four samples. None of the tumors were homogeneously diploid. These results illustrate the importance of multiple biopsy samples in the evaluation of the DNA pattern in colorectal tumors.     

Liquid-based vs. conventional smears in fine needle aspiration of bone and soft tissue tumors

OBJECTIVE: To compare the accuracy of fine needle aspiration cytology of bone and soft tissue tumors utilizing ThinPrep (TP) (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) vs. conventional smears (CS). STUDY DESIGN: Fine needle aspiration cytology from bone and soft tissue tumors was processed and assessed for cellularity, nuclear and cytoplasmic preservation, cellular architecture and stromal background with both the TP liquid-based smear technique and conventional methods. RESULTS: An accurate diagnosis was made in 13% of TP cases as compared to 64% in CS cases. CONCLUSION: CS of fine needle aspiration sample is far superior to TP in diagnosing tumors of bone and soft tissues. Preservation of cytoplasmic features and cellular architecture was superior in conventionally prepared smears.     

Diagnosis of clinically unsuspected extrapulmonary tuberculosis by fine needle aspiration: a case report.

BACKGROUND: Mycobacterium tuberculosis (MTb) infection remains the cause of higher morbidity and mortality than any other infectious disease in the world. Intact cellular immunity is necessary to resist the disease, and therefore the AIDS epidemic has greatly contributed to the resurgence of MTb. Depending on the degree of immunosuppression, the presentation of MTb in patients with AIDS can be atypical and difficult to diagnose as compared to the classical presentation of MTb in the nonimmunocompromised population. CASE: A patient who was not known to be HIV positive had a clinical picture of extensive abdominal and pelvic lymphadenopathy without chest radiographic abnormalities. The diagnosis of MTb was made by fine needle aspiration (FNA) of a pelvic lymph node. CONCLUSION: Miliary tuberculosis associated with AIDS may have an unusual clinical presentation and unusual cytologic features on ENA.     

Crystals of alveolar soft part sarcoma in a fine needle aspiration biopsy cytology smear. A case report

BACKGROUND: Alveolar soft part sarcoma (ASPS) is a rare soft tissue tumor. It has characteristic histomorphology, with typical ultrastructural features demonstrating unique crystalloids. It occurs predominantly in adolescents and young adults, in whom the most common location is within the fascial planes of skeletal muscle of the lower extremity. CASE: We present fine needle aspiration biopsy (FNAB) findings along with histopathologic features and ultrastructural appearance of a large gluteal mass in a 29-year-old female. FNAB cytology smears showed single and small groups of polyhedral malignant cells with granular cytoplasm, anisokaryosis and prominent nucleoli. The delicate cytoplasm had a tendency to rupture, with the presence of many bare nuclei. The characteristic crystals were observed in Papanicolaou-stained smears within the cytoplasm and in the background near the tumor cells. This consolidated the radiologic suspicion of ASPS and facilitated the application of relevant ancillary tests. Biopsy of the mass showed the characteristic histologic pattern. Electron microscopy confirmed the diagnosis with demonstration of membrane-bound, rhomboid crystalloids with a latticelike ultrastructure. CONCLUSION: Detection of characteristic crystalloids in Papanicolaou-stained FNAB smears facilitated a proper evaluation and correct diagnosis of ASPS.