Value of lymphocyte marker studies in diagnostic cytopathology

The separation of lymphoreticular neoplasms from poorly differentiated epithelial and mesenchymal tumors and reactive processes can be a difficult problem in cytopathology. Immunodiagnostic techniques can be applied to cytologic specimens to detect cellular antigens, which may aid in their proper identification. We have reviewed 67 cytologic specimens in which immunoperoxidase techniques were employed using antibodies to common leukocyte antigen (HLE1), B-cell markers (B1, Leu 12 and kappa and lambda light chains), T-cell markers (Leu 1, OKT11, Leu 12 and kappa and lambda light chains), T-cell markers (Leu 1, OKT11, OKT4 and OKT8) and monocytes (OKM1 and LeuM1). These specimens included 33 body cavity fluids (21 pleural, 8 ascitic and 4 pericardial), 22 cerebrospinal fluids (CSF) and 12 fine needle aspirates (4 brain, 1 adrenal, 2 liver, 1 kidney, 3 retroperitoneal masses and 1 lymph node). The marker studies confirmed the initial cytomorphologic diagnoses in 31 specimens and modified the final diagnoses in 16 specimens. Markers in 20 specimens were noncontributory due to low cellularity or technical difficulties. Two problems may limit the usefulness of these procedures. First, many of the CSF specimens contained too few cells for adequate processing. Second, the mesothelial cells from pleural specimens often stained with HLE1. Our findings indicate that marker studies are of value in the diagnosis of problematic cases presenting as undifferentiated tumors in cytopathology.     

Immunocytochemical characterization of large-cell carcinomas of the lung. Role, limitations and technical considerations.

To clarify the use of cytologic preparations, particularly those previously stained by the Papanicolaou method, for the immunocytochemical evaluation of large-cell carcinomas (LCCs), 37 cytologic preparations from cases diagnosed as LCC were examined using a battery of immunocytochemical stains for keratin, chromogranin, common leukocyte antigen (CLA) and B72.3. Thirty-two specimens were from the thoracopulmonary region (12 fine needle aspirates of the lung, 7 bronchial brushings, 5 bronchial washings, 2 sputa and 6 pleural fluids); the remaining specimens were fine needle aspirates of 3 lymph nodes, 1 vertebral body and 1 liver. Of the specimens analyzed, 30 of 37 were positive for keratin and 7 of 35 were positive for B72.3 (6 were positive for both). Only 1 of 37 was positive for CLA while none of 37 was positive for chromogranin. Six specimens showed no reaction with either keratin, B72.3 or chromogranin. These results confirm that the majority of LCCs consist of epithelial cells of either a squamous or an adenocarcinomatous type. They also show that immunocytochemistry is a useful diagnostic adjunct that can be applied to cytologic preparations previously stained by the Papanicolaou method; this is important since immunostaining is often considered after undifferentiated malignant cells are encountered in a previously stained preparation. However, a thorough understanding of some technical limitations is critical in the evaluation of the results of this technique when it is applied to cytologic specimens.     

Use of immunocytochemistry on urinary sediments for the rapid identification of human polyomavirus infection. A case report

The application of immunocytochemistry in urinary cytology for the identification of a human polyomavirus infection is described. The Papanicolaoustained slides of voided urine specimens of a 26-year-old man undergoing steroid therapy showed many inclusion-bearing epithelial cells. After subsequent destaining of the same slides, the presence of human papovavirus antigen in the nuclei of infected exfoliated cells was demonstrated by immunocytochemical staining with simian virus 40 antiserum and peroxidase. Papovaviruses were also detected by electron microscopic study of the smears, confirming the diagnosis of a human polyomavirus infection. The use of immunoperoxidase studies proved to be advantageous for the rapid cytodiagnosis of human polyomavirus infection in the urinary specimens in this case; such studies may be of particular value in equivocal cases to prove or disprove the viral nature of morphologic changes observed in routine preparations.     

Myeloma with involvement of the serous cavities. Cytologic and immunochemical diagnosis and literature review

The significance of serous cavity involvement by myeloma was evaluated in two patients with pleural involvement and two with peritoneal involvement. The involvement occurred at presentation in two patients and after the diagnosis of myeloma in two. The effusions were bloody exudates containing numerous atypical plasma cells. The diagnosis of cavitary involvement was made by morphologic examination of air-dried smears of the effusions, supplemented by the immunocytochemical demonstration of monoclonal proliferation of the plasma cells. In all four cases, these cells contained cytoplasmic kappa light chain immunoglobulins; many of them also stained positively for epithelial membrane antigen. It was best to interpret these immunocytochemical findings with those of the morphologic and additional immunocytochemical studies; the best results for studies for cytoplasmic immunoglobulins were obtained only if the cells in the effusions were washed before they were used for smear preparations and staining. The four patients responded poorly to treatment, dying 12 days, 16 months, 1 month and 10 days after cavitary involvement was recognized. Review of the literature confirmed that the findings in these cases were similar to those in other cases. Cavitary involvement by myeloma carries an ominous prognosis; an accurate recognition of the plasma cells by morphologic and immunocytochemical studies provides the best method of diagnosing cavitary involvement of myeloma and of predicting the poor outcome in such patients.     

Immunocytochemical Staining of Serous Effusions With the Monoclonal Antibody Ber-Ep4

Cytospin preparations were made from 102 serous effusions for immunocytochemical staining using a panel of monoclonal antibodies including a new monoclonal antibody Ber-EP4. On cytological examination, 32 fluids were reported to contain tumour cells consistent with metastatic adenocarcinoma; 66 contained benign cells only and three were reported to contain cells suspicious of malignancy. One effusion contained tumour cells consistent with malignant mesothelioma. Positive staining of the tumour cells with Ber-EP4 was observed in the 32 effusions (100%) which contained adenocarcinoma cells. No staining of the mesothelial cells in these 32 specimens was observed. Carcinoembryonic antigen, epithelial membrane antigen Ca2 and CD15 staining of tumour cells was noted in 53%, 50%, 50% and 9% of these cases, respectively. None of the mesothelial cells in the benign effusions stained with Ber-EP4. Nor did the malignant mesothelial cells in the only case of malignant mesothelioma. These findings suggest that Ber-EP4 is a valuable addition to antibodies available for the differential diagnosis of mesothelial cells and adenocarcinoma cells in serous effusions.     

Large-volume cytocentrifuge for processing alcohol-fixed cytologic specimens. Application in urinary cytology

The use of a large-volume cytocentrifuge for the routine processing of urine specimens was investigated. Most specimens arrived in the laboratory mixed with 50% ethanol. Polyethylene glycol was added to the fixative supplied to the clinicians or to the cell suspension received in the laboratory. The slides used in the cytocentrifuge were coated with gelatin or poly-L-lysine to minimize cell loss. The resulting monolayers of cells, with occasional true tissue fragments, were stained by the Papanicolaou method. When indicated, immunocytochemical and histochemical stains were used. The technique gave good morphologic details, good cell yield and consistent staining quality. This method has also been applied to other cytologic specimens, such as serous fluids and fine needle aspirates, with good results. The method saves the time of cytotechnologists, and slides prepared by this technique are suitable for automatic staining.     

Immunocytochemistry on fine needle aspirates in paraffin miniblocks

A simplified method of processing of fine needle aspirates for paraffin miniblocks suitable for both morphologic and immunocytochemical evaluation is described. Aspirates were fixed in ethanol at 4 degrees C, dehydrated in acetone and xylene and embedded in paraffin (58 degrees C). All steps were carried out in a single Eppendorf centrifuge tube; the total process took less than four hours. Deparaffinized sections were stained using the alkaline phosphatase-antialkaline phosphatase technique with monoclonal and conventional antibodies helpful in the differential cytologic diagnosis of alcohol-fixed aspiration biopsy specimens. Antibodies to keratin, vimentin, desmin, neurofilaments, glial fibrillary acidic protein, leukocyte-common antigen, synaptophysin and immunoglobulin kappa and lambda light chains reacted positively on the miniblock material. Since the paraffin miniblocks combine the histologic pattern of the tumor with the differentiation-specific information provided by immunocytochemistry, their use can improve the accuracy of tumor typing in aspirates.     

The cytopathology of bone marrow transplantation

Bone marrow transplantation (BMT) is an increasingly effective treatment for patients with hematologic disorders and malignant neoplasms. From 1975 to 1986, 1,457 specimens were obtained for cytologic evaluation from 328 of the 635 patients who received BMTs at Memorial Sloan-Kettering Cancer Center. These specimens consisted of 1,049 cerebrospinal fluids (CSFs) from 265 patients, 292 bronchoscopy specimens from 92 symptomatic patients and 116 other specimens (including brushings from the liver and gastrointestinal tract, sputa, urines and cervico-vaginal smears). CSF specimens examined before and after BMT from 80 (30%) patients showed an increased number of benign, nonepithelial cells, which were mainly lymphocytic or histiocytic in origin. Malignant cells were detected in CSF specimens from 44 (17%) patients. Bronchoscopy specimens from 3 patients had suspicious cells present; those from 27 patients contained opportunistic organisms. Atypical epithelial or lymphoreticular cells were seen in bronchial specimens from 49 patients. All cytologic findings were correlated with the pertinent clinical information as well as biopsy and autopsy material, including histopathologic evidence of graft-versus-host disease. Cytologic evaluation, especially of bronchial and CSF specimens, was useful in diagnosing the presence of malignant neoplasms, infectious organisms, inflammatory responses, reactive lesions and cellular atypia due to treatment.     

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.     

Bronchial cytology in pleural mesothelioma. A report of 3 positive cases, including 1 diagnosed initially on bronchial brushings

OBJECTIVE: To evaluate retrospectively the value of bronchial aspiration cytology in patients with histologically proven pleural mesothelioma, reappraising positive smears in light of conventional microscopic features and, when feasible, immunocytochemical investigations. STUDY DESIGN: In 3 cases of mesothelioma with bronchial brushings positive for malignant cells, the cytologic features were correlated with the histologic findings. RESULTS: Salient microscopic features included scant to moderate cellularity arranged in micropapillary clusters, morular aggregates with scalloped borders and isolated malignant cells. Intercellular clear spaces or windows suggesting a brush border on cell membranes were also noted. In cases with available material, immunocytochemistry was positive for keratins, epithelial membrane antigen and calretinin and negative for carcinoembryonic antigen. All the cases were histologically confirmed epithelial mesotheliomas. CONCLUSION: In rare cases, pleural mesothelioma cells are shed within the airway lumina and can be detected in bronchoscopic cytology specimens. Cytologic features seem comparable to their analogues in pleural effusions. Although no single criterion appears diagnostic, their combined documentation could ensure correct interpretation, especially if supported by a limited immunocytochemical panel.     

Diagnosis of T-cell lymphoma in body fluids: cytologic features and unusual flow cytometric findings

OBJECTIVE: To demonstrate the unique morphologic and phenotypic features observed in cases of T-cell lymphomas presenting as effusions. STUDY DESIGN: Cytologic slides and flow cytometric histograms of 8 cases of body fluids with T-cell lymphoma were retrospectively reviewed. Morphologic features, flow cytometric histograms and immunophenotypes of the cells were evaluated. RESULTS: Three of the 8 cases showed 1 or more of the following: intermediate-to-large cells with an increased nuclear-cytoplasmic ratio, finely granular or vacuolated cytoplasm and round or convoluted vesicular nuclei with a prominent single or multiple nucleoli. Flow cytometric studies of these 3 cases showed an abnormal scatter pattern in the myelomonocytic region of the histograms. Phenotypic analysis revealed variable expression of a T-cell phenotype. The remaining cases showed the conventional morphologic and flow cytometric features of a T-cell lymphoma. CONCLUSION: Morphologic alterations of neoplastic T-cells in body fluids can result in a variety of potentially incorrect diagnoses. The unusual flow cytometric histogram can serve as a useful clue for the diagnosis of T-cell lymphoma in body fluids but could be a potential pitfall for a false negative. Detailed cytologic evaluation combined with flow cytometric study can improve diagnostic accuracy.     

Monoclonal antibodies in the cytodiagnosis of serous effusions

In order to assess the value of immunocytochemical staining as a method of discriminating between benign reactive mesothelial cells and malignant epithelial cells in serous effusions, we have studied the reactions of a panel of commercially available antibodies on cells harvested from 83 pleural and peritoneal fluids and compared the results with the clinical and cytological diagnoses. The antibodies used were raised against cytokeratin (PKK1), epithelial membrane antigen (EMA), carcino-embryonic antigen (CEA), pregnancy specific B1-glycoprotein (SP1) and leucocyte common antigen (LCA). Anti-CEA was positive in 16 of 39 effusions (41%) containing carcinoma cells. Pregnancy specific B1-glycoprotein (SP1) was positive in 33% of the same samples. Mesothelial cells did not stain with these antibodies. Thus anti-CEA and SP1 can be used to discriminate between benign mesothelial and malignant epithelial cells in effusions. Anti-PKK1 stained both benign reactive mesothelial cells and malignant epithelial cells and cannot be used to discriminate between these two cell types. Strong positive staining of malignant cells was noted with anti-EMA. However, as occasional weak staining of mesothelial cells was also noted, strong staining with this antibody may be regarded as suspicious but not conclusive of malignancy.     

An immunocytochemical study of normal and abnormal human cerebrospinal fluid with monoclonal antibodies to glial fibrillary acidic protein

The presence of astrocytes in the cerebrospinal fluid (CSF) of patients may be of diagnostic importance. However, the frequency with which astrocytes are shed into normal and abnormal human CSF is unknown. This issue was studied using monoclonal antibodies to an astrocyte-specific antigen, glial fibrillary acidic protein (GFAP), and immunoperoxidase cytochemistry. The study was prospectively conducted on 108 CSF preparations diagnosed as normal, reactive, metastatic malignancy or suspicious for metastatic malignancy. To validate these methods, cells from a clonal human glioma cell line, which contains astrocytes rich in GFAP, were processed in a manner identical to that used for the CSFs obtained from patients. Studies of the human glioma cell line demonstrated intense GFAP immunoreactivity in the majority of the malignant astrocytes. In contrast, none of the CSFs contained GFAP-positive cells. We conclude that immunocytochemical methods can detect GFAP in neoplastic human astrocytes but that nonneoplastic GFAP-positive cells are uncommon in human CSF; such cells were not seen in our large series of normal and abnormal human CSFs. The immunocytochemical detection of GFAP may be a useful criterion for distinguishing malignant astrocytes from other types of malignant cells in human CSF.     

Comparison of cellular phenotypes in tumor cyst and ascitic fluid from ovarian serous carcinoma.

Density gradient centrifugation was applied to isolate cell subsets from tumor cyst and ascitic fluid in eight patients with ovarian serous carcinoma. A comparison of cellular composition and immunologic reactivity of cells from the cysts and from ascitic fluid in each patient was performed. Some differences in density profiles were found, but in each case the consistency of morphologic cell forms in the primary tumor and ascites was documented. Immunophenotypic analyses of isolated cellular fractions using polyclonal and monoclonal antibodies against ovarian carcinoma-associated antigens showed significant immunologic intratumoral heterogeneity. However, there was a similarity of antigen expression in cells from the primary tumors and ascitic fluids. Our study indicated that morphologic and antigenic characterization of a given tumor could be determined in a single representative sample of ascitic fluid.     

Mixed osteoclastic/pleomorphic giant cell tumor of the pancreas: a case report

BACKGROUND: Mixed giant cell tumor (MGCT) of the pancreas is a rare malignant neoplasm. The tumor contains pleomorphic giant cells (PGC), pleomorphic mononuclear cells (PMC) and osteoclastic giant cells (OGC). We describe the first fine needle aspiration biopsy (FNAB) diagnosis of this tumor. CASE: A 76-year-old woman was discovered (on imaging studies) to have an apparently inoperable mass in the head of the pancreas. Computed tomography-guided FNAB showed a malignant neoplasm with features of an MGCT. PGC/PMC, OGC and spindle cells were present. The PGC/PMC expressed epithelial antigens, pancytokeratin, CAM 5.2, AE1/AE3 and epithelial membrane antigen (EMA). The spindle cells focally stained for EMA. OGC were negative for the epithelial antigens. OGC, PGC/PMC and the spindle cells were positive for the mesenchymal marker vimentin. CONCLUSION: FNAB was instrumental in making the diagnosis of a rare pancreatic tumor, MGCT. Immunocytochemistry was helpful in making a definitive diagnosis and suggested that MGCT is a carcinosarcoma like neoplasm. The morphology and immunocytochemical profile raise the possibility that osteoclastic giant cell tumor and pleomorphic giant cell tumor may be different morphologic and biologic expressions of the same tumor.     

Peroxidase-labeled antibody staining for carcinoembryonic antigen of cytologic specimens for light and electron microscopy

Immunohistochemical staining methods suitable for light and electron microscopic examination of cytologic specimens are described. Application of the methods clearly demonstrated the localization of carcinoembryonic antigen (CEA) in adenocarcinoma cells in body fluids. The use of a peroxidase-labeled antibody method permits rapid penetration of the cells by the antibody, which is not achieved by the peroxidase-antiperoxidase or avidin-biotin-peroxidase-complex staining methods. Since mesothelial and inflammatory cells are negative for CEA, the staining of body fluids for CEA is expected to be an extremely useful tool for the differential diagnosis of adenocarcinoma.     

Evaluation of an antibody to human milk fat globule antigen in the detection of metastatic carcinoma in pleural, pericardial and peritoneal fluids

An antibody to human milk fat globule-2 (HMFG-2) antigen was investigated to assess its value in detecting tumors in pleural, pericardial and peritoneal fluids. One hundred forty consecutive fluids were evaluated using the avidin-biotin complex (ABC) technique. Conventional cytology and HMFG-2-stained smears were compared using the former as the standard for tumor detection. Discrepant results were found in 15 specimens (8 false negatives and 7 false positives). Causes for discrepancy between the methods included lack of HMFG-2 antigen on tumor cells, random sampling error and endometrial cells ectopically located in the pelvic cavity or introduced during uterine instrumentation. We conclude staining for HMFG-2 may be a useful adjuvant technique for the detection of rare tumor cells in body fluids provided there is a high index of suspicion of metastasis from an HMFG-2-positive primary neoplasm.     

Tumor marker studies of cervical smears. Potential for automation

The distribution of three tumor markers, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and prekeratin (PK) was studied in exfoliated epithelial cells in cervical smears using an immunoalkaline phosphatase staining technique to demonstrate the antigens. EMA was expressed by abnormal cells in a consistent and reproducible fashion whereas the other two markers were variably expressed by both normal and abnormal cells. Our results suggest that immunocytochemical staining for EMA could be of value not only for the diagnosis of cervical intraepithelial neoplasia but also for the automated screening of cervical smears.     

Truncated cystatin C in cerebrospiral fluid: Technical [corrected] artefact or biological process?

Cystatin C, a low molecular weight cysteine proteinase inhibitor present in human body fluids at physiological concentrations, is more expressed in cerebrospinal fluid (CSF) than in plasma. Mass spectrometric characterization showed that after 3 months of storage of human CSF at -20 degrees C, cystatin C was cleaved in the peptide bond between R8 and L9 and lost its eight N-termini amino acids, whereas this cleavage did not occur when stored at -80 degrees C. This truncation occurred in all CSF samples studied irrespective of the underlying neurological status, indicating a storage-related artefact rather than a physiological or pathological processing of the protein. These results stress the importance of optimal preanalytical storage conditions of any sample prior to proteomics studies.     

Preclinical feasibility study of NMP179, a nuclear matrix protein marker for cervical dysplasia

OBJECTIVE: To evaluate, in a preclinical feasibility study, the efficacy of NMP179, a monoclonal antibody recognizing a cervical tumor-associated nuclear matrix antigen, for the early detection of high and low grade cervical intraepithelial neoplasia. STUDY DESIGN: In a blind study involving two clinical sites, NMP179 immunocytochemical staining data from 261 cervicovaginal Thin-Prep specimens were evaluated. Assay sensitivity and specificity were calculated based upon a positive threshold of > 10 immunostained cells per case, using cytologic diagnosis as an end point. RESULTS: Based upon the examination of squamous epithelial cells, NMP179 detected 96.7% of cases with cytologically diagnosed high grade squamous intraepithelial lesions (HSIL) and 70.5% of low grade squamous intraepithelial lesions. The antibody also reacted with 29.6% of normal (within normal limits or benign cellular changes) smears. CONCLUSION: The NMP179 assay detected HSIL with very high accuracy (96.7%). The assay was 79.3% sensitive for the detection of low and high grade cervical intraepithelial neoplasia (grades 1-3), with a specificity of 70.4%. NMP179 may be an effective marker for the early detection of preneoplastic squamous intraepithelial lesions of the cervix and may be useful as an adjunctive tool for better management of cervical intraepithelial neoplasia.