Endogenous phosphatase activity in tumor cells in a liver aspirate. A potential problem for immunocytodiagnosis using immunoalkaline phosphatase methods

Immunocytochemical studies were performed on fine needle aspirates of the liver in a patient with hepatocellular carcinoma. A panel of commercially available antibodies was used to study the aspirated cells by immunoalkaline phosphatase and immunoperoxidase methods. The malignant cells in the aspirates, which were positively stained by the immunoperoxidase method for alphafetoprotein and by both methods for epithelial membrane antigen, were most probably hepatocellular in origin. Some cells were shown by the immunoalkaline phosphatase method to possess leukocyte-common antigen (LCA) and antigens of colonic and ovarian tissues. These findings were further investigated, and it was found that the tumor cells indeed had LCA as well as levamisole-resistant alkaline phosphatase activity. Although the immunoalkaline phosphatase methods are useful immunodiagnostic techniques applicable to fine needle aspirates, the endogenous enzyme activity present in some nonhematopoietic tumor cells is a cause for caution in the use of these methods in aspirates from nonhematopoietic tumor tissues.     

Immunocytochemistry of cerebrospinal fluid

In order to determine how best to study cells in cerebrospinal fluid (CSF) by immunocytochemical techniques, several crucial technical variables and five immunocytochemical methods were examined. Immunocytochemical studies could be performed on either cell suspensions or smears. The method using cell suspensions was more sensitive, producing less background staining, but requiring more cells than that using smears. Among the five methods examined, indirect immunoperoxidase (IP) and indirect immunoalkaline phosphatase (IAP) were comparable in sensitivity. The peroxidase-antiperoxidase (PAP), alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin complex-immunoalkaline phosphatase (ABC-AP) methods were comparable in sensitivity and were more sensitive than either the IP or IAP technique. The peroxidase methods were plagued with problems related to endogenous enzyme activity and the ABC-AP method may exhibit undesirable background staining. Therefore, the IAP method should be used for cell suspensions and the APAAP for cells on smears. In CSF specimens with a small number of cells, immunocytochemical studies should be done on smears by the APAAP method. These conclusions are supported by our experience with CSF specimens from patients with reactive and neoplastic lymphocytoses.     

Parotid metastasis of Merkel cell carcinoma in a young patient with ectodermal dysplasia. Diagnosis by fine needle aspiration cytology and immunocytochemistry.

Fine needle aspiration (FNA) biopsy was performed on an intraparotid lymph node metastasis of a Merkel cell carcinoma of the eyelid in a 15-year-old girl with antecedent ectodermal dysplasia syndrome. The cytologic appearance of the aspirate and the results of immunocytochemical typing of intermediate filaments on the FNA smears provided a definitive diagnosis. The Romanowsky stain provided an excellent delineation of paranuclear intracytoplasmic "buttons," which appeared to contain both cytokeratin and neurofilaments by immunocytochemical studies. These findings confirm previous data emphasizing the role of light microscopic observations, supplemented by proper immunocytochemical investigations, in the differential diagnosis of metastatic Merkel cell carcinoma in fine needle aspirates.     

Fine needle aspiration cytology of metastatic malignant schwannoma. A case report

A metastatic malignant schwannoma diagnosed by fine needle aspiration (FNA) biopsy in a 56-year-old man is reported. Cytologic examination of smears and cell blocks prepared from aspirates of a vertebral mass suggested the presence of metastases from a previously excised malignant schwannoma on the right leg. Electron microscopic and immunocytochemical studies on the aspirate supported the diagnosis, as did the patient's clinical history and previous pathology and the radiographic demonstration of metastatic lesions in the lung. The cytologic findings (cells with oval-to-spindled nuclei and ill-defined cellular borders suspended within a delicately fibrillar eosinophilic matrix) are discussed in light of the histologic diversity of this lesion and the problems of distinguishing it from other sarcomas. The ability to diagnose metastatic malignant schwannoma by FNA emphasizes the value of this technique.     

Morphologic and immunocytochemical evaluation of 220 fine needle aspirates of malignant lymphoma and lymphoid hyperplasia

A total of 220 fine needle aspiration (FNA) specimens from 212 patients with clinically suspected or previously histologically confirmed lymphoma were evaluated by cytology in conjunction with immunophenotyping analysis of the aspirate; the results were compared with the histologic diagnosis made on previous or current accessions of lymph node or extranodal tissue. Smears of the aspirates were stained with the Diff-Quik and Papanicolaou stains while immunoperoxidase staining using antibodies against kappa and lambda immunoglobulin light chains and Leu-4 was routinely performed on Cytospin preparations. Where indicated, additional marker studies (including T-200, Leu-1, Leu-2a, Leu-3a + 3b, Leu-M1, B1, Leu-12, IgM, CALLA and TdT) were performed. For the non-Hodgkin's lymphomas, specimens were classified by the cytologic characteristics of the neoplastic cells according to the International Working Formulation scheme. The combination of cytologic smears and immunoperoxidase studies resulted in a diagnosis of lymphoma in 173 cases (79%). The remaining aspirates were interpreted as suspicious for lymphoma (7%), benign (10%) or inadequate for diagnosis (4%). Of the 15 suspicious aspirates, 5 proved to be Hodgkin's disease and 2 to be T-cell lymphoma by subsequent biopsy. The cause of failure in the nine inadequate aspirates were necrosis (3 cases), sclerosis (2 cases) and faulty technique (4 cases). In the cases that had concurrent tissue biopsies, no false-positive diagnoses were rendered. These results indicate that FNA used in association with immunocytochemistry is a reliable tool for establishing the diagnosis and classification of the majority of cases of lymphoma. Optimal immunoglobulin light-chain ratios for defining monoclonality in FNA specimens of B-cell lymphomas are proposed.     

Fine needle aspiration cytology and immunocytochemistry in a case of intrathoracic thyroid goiter

Fine needle aspiration (FNA) was performed in the case of a patient with an anterior mediastinal mass. Examination of the smears revealed individual and groups of benign nondiagnostic cells. Surgical removal and histologic examination indicated that the mass was a true intrathoracic thyroid goiter. Subsequent immunocytochemical studies on the FNA smears showed thyroglobulin in the cytoplasm of the aspirated cells. The cytologic findings are presented; while not diagnostic of a thyroidal origin in this case, they serve as a reminder of the wide range of cytologic appearances of colloid nodules and goiters. This case will hopefully heighten the awareness of cytologists and other physicians to the consideration of the possibility of intrathoracic goiter in the differential diagnosis of mediastinal lesions seen in fine needle aspirates.     

Fine needle aspiration biopsy of peripheral congenital bronchial cyst

Bronchial cysts usually occur centrally near the mediastinum, but may present as a peripheral "coin" lesion requiring distinction from other causes of coin lesions of the lung. A case is reported in which fine needle aspiration (FNA) biopsy was used to make the diagnosis of such a peripheral bronchial cyst. FNA produced an opaque opalescent fluid; cytologic study of the smears showed abundant bipolar birefringent needle-shaped crystals an a few macrophages. Neither special staining of some smears nor cultures of part of the aspirate revealed the presence of any organism. The cytologic diagnosis was confirmed by histologic examination of the subsequent resection specimen.     

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.     

Cytodiagnosis of extrapleural tumors in multiple myeloma by fine needle aspiration. A report of three cases.

Cytologic and immunocytochemical studies were done on fine needle aspirates of extrapleural tumors from three patients with multiple myeloma. In the first case the cytologic findings were consistent with myeloma, but the immunochemical studies were done improperly and were not interpretable. In the second case the cytologic findings were those of acute inflammation despite the strong clinical suspicion of the thoracic lesion's malignancy. In the third case both the cytologic and immunocytochemical findings were diagnostic of plasmacytoma. Our experience with these cases suggests that an assessment of the nature of the extrapleural tumor is indicated even in patients with known myeloma, that fine needle aspiration (FNA) is the diagnostic procedure of choice under the circumstances and that immunocytochemical studies can enhance the diagnostic accuracy of FNA for plasmacytoma. The best results for assessment of the cytologic FNA findings in extrapleural tumor in myeloma can be achieved by proper execution of both the FNA procedure and immunochemical studies and by interpretation of the cytologic findings within the context of the clinical findings.     

Immunocytochemical diagnosis of lymphoma in serous effusions

An immunoalkaline phosphatase technique was used to examine the lymphoid cells in serous effusions from five patients with malignant lymphoma. The results were interpreted along with the morphologic studies and retrospective assessments of the clinical conditions of the patients. Two patients had no involvement of the serous cavities, and two had proven involvement. The fifth patient was studied while his lymphoma was evolving from inapparent to disseminated disease. In the two patients without involvement of the serous cavities, the effusion lymphocytes were predominantly monoclonal T cells, comparable to those in six patients with diseases other than lymphoma. In those with involvement of the serous cavities, the effusion lymphocytes were predominantly monoclonal B cells. In the patient with lymphoma in evolution, immunocytochemical studies accurately reflected the progression of disease. We conclude that immunocytochemical studies of the lymphocytes in serous effusions help not only to differentiate reactive from neoplastic lymphoproliferation but also to assess the status of lymphomatous involvement of the serous cavities. The immunocytochemical studies are most effective when correlated with clinical and cytologic studies.     

Fine needle aspiration cytology of focal liver lesions. Results obtained with examination of both cytologic and histologic preparations

The results of 197 consecutive fine needle aspirations (FNA) of focal liver lesions performed on 176 patients were reviewed, and the 176 single most diagnostic aspirates were analyzed in detail. The majority of specimens were obtained using a 20-gauge or 22-gauge needle with ultrasound guidance. An attempt was made to obtain both a cytologic and a tissue specimen from each aspirate. The overall accuracy of the procedure was 85%; the accuracies of the tissue and cytologic specimens were 67% and 73%, respectively. The combined procedure detected 81% of the documented malignant tumors; the tissue specimen detected 62%. Eleven tumors were identified only in the tissue specimen and 23 were identified only in the cytologic specimen. There were no false-positive diagnoses. Six of nine hepatocellular carcinomas were detected. These results show that FNA cytology is a safe, accurate, relatively noninvasive technique whose diagnostic yield may be improved by examination of both a histologic tissue and a cytologic preparation.     

Primary multilobated T-cell lymphoma of the breast diagnosed by fine needle aspiration cytology and immunocytochemistry

The fine needle aspiration (FNA) cytologic, immunocytochemical and ultrastructural findings of a primary multilobated T-cell lymphoma arising in the breast of a 61-year-old woman are described. Large pleomorphic multilobated malignant cells were primarily identified as lymphomatous in origin and phenotypically as T-cells by a selected panel of monoclonal antibodies applied to the original smears obtained by FNA biopsy. This appears to be the second report of a multilobated lymphoma arising in the breast and the first with a T-cell phenotype in this anatomic site.     

Fine needle aspiration biopsy of primary malignant fibrous histiocytoma of the lung

Malignant cells were found on the fine needle aspiration (FNA) biopsy smears from a primary tumor of the lung. The cytologic specimen contained a mixture of fibroblastlike cells, histiocytelike cells, giant cells and undifferentiated cells; mitotic figures were also found. Histologic, histochemical and electron microscopic studies confirmed the FNA cytologic suggestion of a primary malignant fibrous histiocytoma of the lung.     

C-Erbb-2 Expression and Dna Ploidy Status In Breast Cancer Cells Obtained By Fine Needle Aspiration (Fna)

The aim of this study was to determine the relative rate of c-erbB-2 oncoprotein immuno-detection on matched fine needle aspiration (FNA) smears and surgical specimens of breast cancer, and to correlate the c-erbB-2 expression with the assessment of the DNA ploidy status. the expression of c-erbB-2 oncoprotein was evaluated using an immunoalkaline phosphatase technique in 49 breast aspirates (four benign and 45 malignant lesions) and 21 matched surgical specimens. the DNA ploidy status was assessed by densitometric techniques on Feulgen-stained smears. Fifty-eight per cent of the smears obtained from 45 malignant lesions and 43% of the 21 corresponding paraffin sections contained cells that were stained by the antibody. the higher incidence of c-erbB-2 expression on smears seems to be due mainly to the better antigen preservation in the fresh cytological preparations. the correlation between c-erbB-2 oncogene expression and DNA ploidy assessment showed an increased incidence of oncogene expression in aneuploid tumours (71% vs 29%; P > 0.05).     

Fine needle aspiration diagnosis of malignant lymphoma with confirmation by immunoperoxidase staining

Cytospin preparations of fine needle aspirates in 14 cases of suspected lymphoma were studied by immunoperoxidase techniques. The combination of cytologic smears and immunoperoxidase studies resulted in a working diagnosis in 13 of the 14 cases. The immunologic markers in conjunction with the cytologic appearance of the aspirates were reliable and consistent in differentiating between malignant and benign lymphoproliferative lesions and in determining the B-cell or T-cell nature of the process.     

Myoepithelioma of the parotid gland initially diagnosed by fine needle aspiration cytology and immunocytochemistry: a case report

BACKGROUND: Myoepithelioma is a rare, benign tumor of the salivary gland, most commonly affecting the parotid gland. Although the cytologic features of myoepithelioma are documented in a few case reports, it has rarely been diagnosed preoperatively by fine needle aspiration (FNA) cytology. CASE: A 33-year-old man presented with a left parotid swelling 2.5 cm in diameter and of about 5 years' duration. FNA smears showed bundles of spindle-shaped cells as well as plasmacytoid and stellate cells in sheets and dissociated forms. A few cells had nuclear grooves, and occasional cells showed intranuclear cytoplasmic inclusions. In May-Grünwald-Giemsa-stained smears, most of the cells had reddish cytoplasm. Red to purple, myxoid matrix was present as a scanty fibrillar substance and as globules surrounded by tumor cells vaguely reminiscent of adenoid cystic carcinoma. A cytodiagnosis of myoepithelioma was given and corroborated by immunocytochemical staining, which revealed a positive reaction for vimentin, smooth muscle actin and S-100 protein. Epithelial membrane antigen yielded a negative reaction except for a few plasmacytoid cells with weakly positive staining. Histopathology of the resected tumor and immunohistochemical staining confirmed the cytodiagnosis of myoepithelioma. CONCLUSION: FNA cytologic features together with immunocytochemical studies on smears can offer a preoperative diagnosis of myoepithelioma.     

Fine needle aspiration cytology of neuroblastoma, including peripheral neuroectodermal tumor, with immunocytochemical and ultrastructural confirmation

Eleven fine needle aspiration (FNA) biopsies were performed on seven children with neuroblastoma, including one patient with a congenital neuroblastoma and another with a peripheral neuroblastoma of the thoracopulmonary region. FNA cytology made the primary diagnosis of neuroblastoma in four of the seven cases. The other biopsies documented local recurrences and metastases to liver, lymph nodes, orbit and breast. The cytologic features included varying numbers of small primitive cells with scanty cytoplasm, poorly to well-formed pseudorosettes, cell processes, a fibrillary matrix and multinucleated ganglion cells. Five of the seven patients had electron microscopic (EM) examination of the FNA specimen, which in all cases confirmed the diagnosis. Batteries of immunoperoxidase stains were performed on all 11 aspirates with variable results. Staining for neuron-specific enolase was positive in four of the five neoplasms tested, although strongly positive in only three of the cases. Staining for neurofilament markers was positive in only two of five tumors. Studies for cytokeratin markers (AE1/3), low-molecular-weight cytokeratin (35BH11), hematopoietic markers (T29/33), immunoglobulin light chains and myoglobin were negative. One case was positive for vimentin. This study attests to the value of FNA cytology in suggesting a correct diagnosis of either primary, recurrent or metastatic neuroblastoma in children. Selective use of immunoperoxidase stains and EM on the aspirates may be of value.     

Fine needle aspiration biopsy of the breast. Influence of the number of passes and the sample size on the diagnostic yield

The sensitivity of fine needle aspiration (FNA) biopsy of the breast as a function of the number of aspirations performed on any given lesion was investigated. Four separate aspirations each were performed on over 400 lesions of the breast, 93 of which yielded a cytologic diagnosis. The incremental diagnostic yields of each subsequent aspiration were tabulated. The first aspirate of the sequence gave the greatest yield, with smaller incremental yields on the second through the fourth aspirates. Benign and malignant lesions gave similar results, as did palpable and nonpalpable lesions (the latter being usually smaller in size). A mathematical extrapolation of the data indicates that three or four aspirations of any given lesion provide the optimal yield within the limits of practicality. This performance of multiple FNA biopsies is particularly important when the pathologist does not perform the biopsy, or is unable to assist in the immediate interpretation of the specimen to assess its adequacy.     

Double and triple immunocytochemical labelling at the light microscope level in histopathology

Summary Double and triple immunocytochemical detection methods for routine use in histopathology were investigated. For double immunostaining, the combinations of immunogold-silver staining (IGSS, black) with an immunoperoxidase method (3-amino-9-ethylcarbazole, red-brown) or with an immunoalkaline phosphatase method (Fast Red TR, red) proved very useful. These were followed by a Haematoxylin counterstain. An alternative approach using immunoperoxidase (red-brown) and immunoalkaline phosphatase (Fast Blue, BB, blue) methods was also successful, particularly for frozen sections of unfixed tissue and for the establishment of intermediate filament coexpression in tumours. The coexistence of desmin with vimentin in rhabdomyosarcoma, and of glial fibrillary acidic protein with vimentin in ependymoma, could be demonstrated directly by means of non-crossreacting murine and rabbit antibodies in the above combinations. The black (IGSS), red-brown (immunoperoxidase) and blue )immunoalkaline phosphatase) colours gave excellent contrast in triple immunostaining. The side-by-side demonstration of helper and suppressor T lymphocytes during renal allograft rejection, of kappa and lambda light chains in B-immunoblastic lymphoma, and of T and B lymphocyte populations in non-Hodgkin's lymphomas provided immediate information on the topography and cellular organization of the tissues.     

Preparatory methods for DNA hydrolysis, cytochemistry, immunocytochemistry and ploidy analysis. Their application to automated and routine diagnostic cytopathology

A review is presented of some methods used to prepare cytologic specimens for analytical and/or automated studies, with the steps of the procedures detailed in appendices. The preparation of the cell monolayers required for optimal automated cell image analysis and classification, e.g., by the Cytoscan 110, is discussed, as is the preparation of poly-L-lysine-coated slides used in the production of monolayered specimens. These monolayers, which can be prepared from a variety of specimens, are also useful for cytochemical and immunocytochemical studies and DNA ploidy analysis. For DNA analysis, a modified gallocyanin chrome alum staining procedure is described as a stoichiometric alternative to the time-consuming Feulgen reaction. The hydrolysis technique required by the latter method is also detailed. The freeze-fracturing technique for the enhancement of monoclonal antibody immunocytochemical staining of detectable antigens is described, along with an indirect immunoalkaline phosphatase staining method. The use of enzyme cytochemical reactions for glucose 6 phosphate dehydrogenase and lysosomal naphthylamidase is also presented.