Rapid method for identification of macrophages in suspension by acid alpha-naphthyl acetate esterase activity

A supravital staining procedure for the identification of macrophages in cell suspension using a modification of a standard cytochemical assay for alpha-naphthyl acetate esterase (ANAE) activity is described. Macrophages are stained an intense red-brown after 5 min incubation in a buffer using ANAE as the substrate and hexazonium pararosaniline as the coupler for the azo dye. There is close agreement in the number of ANAE-positive cells found and the number of macrophages identified in smears by morphological criteria, by phagocytosis, and by the presence of Fc receptors. Therefore, this stain provides a quick, inexpensive method to estimate the number of macrophages present in suspensions of lymphocytic tissues from rats and mice.     

Cytology of polychrome-stained equine synovial fluid smears. Comparison with clinical findings, histologic specimens, Wright-Giemsa-stained smears and outcome.

Polychrome-stained equine synovial fluid specimens from 34 normal joints and 129 joints with clinical abnormalities were examined cytologically. The smears from joints with abnormalities were categorized as within normal limits (4.7%), slight abnormality (27.9%), proliferative synovitis (21.7%), neutrophilic pattern (20.2%), elongated cell pattern (10.1%), other moderate to marked abnormality (11.6%) and unsatisfactory (3.9%). Cytologic abnormalities that were not restricted to a single category included spindle cells, crystals, stellate cells and cartilage fragments. Multinucleate cells and mononucleate cells with dense cytoplasm and a delicate periphery were seen in smears from cases with clinical diagnoses of osteochondrosis or fracture; interpretation of these cells as osteoclasts and their mononucleate precursors was supported by positive staining with tartrate-resistant acid phosphatase. Smears within the same cytologic category were not found to correspond with a single clinical diagnosis. The identification of several cytologic patterns in cases with the same clinical diagnosis suggests that multiple stages of disease were sampled. Except in cases with the cytologic neutrophilic pattern, there was not a consistent relationship between the histologic features in synovial biopsy specimens and the cytologic findings; the morphologic variation within synovial membrane sections and between sections from different locations was sometimes marked. When compared with air-dried, Wright-Giemsa-stained smears, the polychrome-stained smears were more sensitive in the detection of cytologic abnormalities and were less often falsely negative or unsatisfactory. Following surgery, cases with clinical diagnoses of osteochondrosis (29 cases) and fracture (25 cases) were analyzed according to clinical outcome and cytologic category. While 80% of the horses with proliferative synovitis in cytologic specimens were sound, only 67% of those with the elongated cell pattern, 50% of those with slight abnormality and 33% of those with other moderate to marked abnormality were sound. A statistically significant relationship (P less than .02) was found in cases with a diagnosis of osteochondrosis: animals with a proliferative synovitis pattern were almost three times as likely to be sound as compared to those with slight abnormality. These findings indicate that polychrome-stained equine synovial fluid smears (1) provide information that is different from that found in corresponding histologic sections and (2) are superior to air-dried, Wright-Giemsa-stained smears for cytologic examination. The polychrome-stained equine synovial fluid smears were found to provide information supportive of clinical, radiographic and prognostic data.     

Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.     

Microfilariae of Wuchereria bancrofti in cytologic smears

Microfilariae of Wuchereria bancrofti were observed in cytologic material in 35 cases. The material included cervicovaginal smears (17 cases), effusions (14), urine (2), bronchial washings (1) and ovarian cyst fluid (1). The initial diagnosis was made from the cytologic smear in all cases; none had clinical filariasis. Symptomatic vaginal bleeding in 9 of the 17 cases with microfilaria-positive cervicovaginal smears was reflected in the large numbers of red blood cells found in the smear. Blood eosinophilia was present in 11 of 19 cases investigated. Eosinophils were seen in the smears in 20 cases. In the majority of the cases of effusions with microfilariae the effusions were malignant. Significant adherence of inflammatory cells and macrophages to microfilariae was present in 7 of the 35 cases. The significance of these findings is discussed.     

Cytology of bronchial benign granular-cell tumor

The cytologic features of a case with multiple bronchial benign granular-cell tumors are reported and compared with those of previously reported cases. Characteristic tumor cells were found in the bronchoscopic brushing smears and in cell block sections (but not smears) prepared from the washing fluid. These findings were confirmed by the bronchial biopsy and histologic study of the resected tumors. A cytologic diagnosis of bronchial granular-cell tumor should not be difficult because the cytologic appearance of the tumor cells is characteristic; however, the possibility of a concomitant tumor, such as adenocarcinoma or small-cell carcinoma, should be considered and excluded.     

Fluoride resistant alpha-naphthyl acetate esterase and combined enzyme histochemistry in the study of normal and pathologic lymphoid tissues

Alpha-Naphthyl acetate esterase (ANAE) and fluoride resistant alpha-naphthyl acetate esterase (FRANAE) have been compared as histochemical methods to identify T lymphocytes in sections of normal and pathological human lymphoid tissues. In addition, the FRANAE method was combined with alkaline phosphatase (ALP) in order to simultaneously evaluate the relationship between T lymphocytes and fibroblastic reticular cells (ALP) positive). The "dot like" esterase positivity of T lymphocyte was better evaluated by using FRANAE when compared to ANAE because of fluoride inhibitor of the strong esterase activity of dendritic cells and most macrophages. The combined ALP-FRANAE method clearly demonstrated a large number of fibroblastic reticular cells within the T-areas in various normal and pathological tissues such as hyperplastic lymph nodes and especially in the lymph nodes and spleens from patients with Hodgkin's disease.     

Histochemical identification of human T lymphocytes from paraffin sections

The alpha-naphthyl acetate esterase (ANEA) is a histochemical marker for human T lymphocytes in cell smears and frozen tissue sections. We have now applied the ANAE method to paraffin-embedded tissue sections. We first demonstrated with cytocentrifuged cell smears of blood leukocytes that the ANAE activity is preserved upon prolonged storage in formol calcium, Holt's buffer, acetone, xylene, and heat. When the tissue sections were similarly processed and embedded in paraffin, the ANAE positive (T) lymphocytes were identified by their distinct display of one or more reddish-brown reaction dots in the cell cytoplasm. ANAE positive mononuclear phagocytes were easioy distinguished from the T lymphocytes by their diffuse, sodium fluoride-sensitive pancytoplasmic reaction. The extension of the ANAE method to paraffin-embedded tissue sections with superior morphological integrity, makes it possible to apply it in practical biopsy pathology.     

Diffuse alveolar damage. Morphologic features in bronchoalveolar lavage fluid

OBJECTIVE: To evaluate the overall cytologic characteristics of diffuse alveolar damage (DAD) in bronchoalveolar lavage (BAL) specimens in search of features that could be useful in cytologic diagnosis. STUDY DESIGN: We evaluated BAL samples from patients with DAD obtained simultaneously with transbronchial biopsies (n = 8) or open lung biopsies (n = 2) or within 24 hours of autopsy (n = 2). The material was processed routinely for cytologic and histologic evaluation. RESULTS: The smears were moderately to highly cellular. All cases had large numbers of alveolar macrophages and/or desquamated alveolar cells. The epithelial component displayed various degrees of nuclear atypia. Some epithelial clusters were three-dimensional, with peripheral cells showing clear cytoplasm, protruding outwards and resembling hobnails. Other aggregates appeared two-dimensional, as sheets of cells with flattened and dense cytoplasm (squamotized). Both types of cell clusters were often associated with dense, basophilic or amphophilic, amorphous extracellular material. Counterparts of all the cytologic features were observed in the histologic material, including atypia of the alveolar lining with hobnailing, squamotization, amorphous extracellular material and hyaline membranes. CONCLUSION: The cytologic features of BAL represent a constellation of alveolar cell injury. Based on these features, DAD can be correctly diagnosed or suggested in BAL samples in the appropriate clinical setting.     

Abrasive bronchial brushing cytology. A preliminary study of 200 specimens for the diagnosis of neoplastic and nonneoplastic bronchopulmonary lesions

Two hundred subjects with chronic respiratory symptoms with a suspicion of malignancy were selected for bronchial brushing cytology. Prior sputum examination had shown malignant squamous cells in two cases only. The cytologic appearances of the brushing smears were divided into five categories: 41 (20.5%) smears with positively malignant cells; 20 (10%) smears predominantly showing chronic inflammatory features; 31 (15.5%) smears with mainly acute inflammatory changes; 60 (30%) smears with normal cytologic features; and 48 (24%) smears unsatisfactory for cytologic interpretation. Thirteen patients with a positive cytology had a positive tissue biopsy for malignancy. Among the group with chronic inflammatory changes, acid-fast bacilli were identified in nine cases, and one smear showed frank tuberculous granuloma. In the unsatisfactory group, two cases showed malignant cells in the postbrushing sputum. There was one false-negative report for malignancy in the entire study. This study confirms the sensitivity and accuracy of bronchial brushing cytology in the diagnosis of various bronchopulmonary lesions, especially malignancy and pulmonary tuberculosis, in India.     

Cytologic study of tissue repair in human bronchial epithelium

The cytologic findings of atypical cells considered to be tissue repair cells after mechanical injury to the bronchial epithelium are reported. These cells were studied in sequential bronchial brushing smears from patients who underwent repeated bronchoscopies for the diagnosis of lung cancer. The cellular findings varied according to the length of time since the previous bronchial brushing. Many cell clusters of highly atypical cells in two-dimensional sheets with large nuclei and prominent nucleoli were observed in specimens taken two or three days after a previous brushing; mitotic figures were observed on day two. In specimens taken on days four and five, the number of atypical cells was decreased and the degree of atypia was slight.     

The effect of delivery on regression of abnormal cervical cytologic findings

The purpose of this study was to determine whether abnormal antepartum cervical cytologic findings result in differing postpartum regression rates. Between 1993 and 2000, 107 pregnant women with antepartum abnormal cervical cytologic findings were identified. Papanicolaou smear data were separated into three groups by use of the CIN classification system. Postpartum regression rates of antepartum Papanicolaou smears were analyzed six months after delivery. Normalization of Papanicolaou smears in the postpartum period were observed in 50 of 107 women (46.7%). Regression of cervical cytologic findings was noted in 61 of 107 women (57%). Respectively, persistence of Papanicolaou smear was noted in 43 of 107 patients (40%). Only 3 of 107 (3%) antepartum findings progressed after delivery. Desquamation of the cervical epithelium or enhancement of a localized reparative immunologic response after vaginal delivery could play an important role in the spontaneous regression of cervical dysplasia in the postpartum period.     

Gynecomastia: cytologic features and diagnostic pitfalls in fine needle aspirates.

OBJECTIVE: To illustrate some of the uncommon cytologic findings of gynecomastia, such as apocrine metaplasia, cellular atypia and foamy macrophages, that can be misinterpreted as evidence of malignancy. STUDY DESIGN: The clinical data and fine needle aspiration (FNA) cytologic material from 100 men with the diagnosis of gynecomastia were retrospectively reviewed. The excisional biopsy slides were available for 16 cases. For comparison, FNA smears from five men with breast lesions other than gynecomastia were studied. RESULTS: The patients ranged in age from 23 to 91 years. Cytologic findings were as follows: cohesive sheets of cells containing 20-1,000 cells (98%); scattered, single, bipolar cells (78%); spindle cells (68%); ductal epithelial atypia (26%); apocrine metaplasia (8%); and foamy histiocytes (12%). In nine cases the atypia was marked, and in two of them the possibility of malignancy could not be ruled out. Surgical follow-up on 16 patients, including the cases with marked atypia, showed gynecomastia. In one case, gynecomastia was associated with intraductal papilloma. No correlation between the underlying etiology and atypical cytologic features of gynecomastia was identified. CONCLUSION: Apocrine metaplasia and epithelial atypia are common findings in gynecomastia. Attention to the cell patterns, the presence of sheets of ductal cells and absence of atypical single cells will point to the correct diagnosis.     

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.     

Semiquantitative assessment of c-erbB-2 (HER-2) status in cytology specimens and tissue sections from breast carcinoma

OBJECTIVE: To determine whether various methods of fixation of surgical pathology specimens from breast carcinomas would influence the outcome of evaluation of the expression levels of c-erbB-2 (HER-2). For this, comparisons were made between (1) alcohol-fixed (95%) and air-dried smears from fresh surgical pathology specimens of breast carcinomas, and (2) formalin-fixed, paraffin-embedded tissue sections of the same specimens. STUDY DESIGN: Alcohol-fixed and air-dried smears or touch preparations were made from 30 fresh mastectomy/lumpectomy surgical pathology specimens from breast carcinomas. Immunohistochemistry was performed using the c-erbB-2 primary antibody against the extracellular domain of the c-erbB-2 gene product. Staining was simultaneously performed on formalin-fixed, paraffin-embedded tissue sections of the same specimens. A semiquantitative approach was used for evaluation of immunostaining by three independent investigators, and a consensus was reached. RESULTS: A total of 30 cases were reviewed. Tissue positivity was determined for c-erbB-2 in: 73% of alcohol-fixed specimens (n = 13 [3+] and n = 9 [2+]), 67% of air-dried smears (n = 9 [+3] and n = 11 [+2]) and 47% (n = 8 [+3]) and n = 6 [+2]) of formalin-fixed, paraffin-embedded tissue specimens. All formalin-fixed tissue specimens that were determined to positively express c-erbB-2 were also found to be positive on the alcohol-fixed smears. CONCLUSION: The incidence of c-erbB-2 expression in fresh cytologic material is significantly higher (P < .05) than in formalin-fixed, paraffin-embedded tissue. Alcohol-fixed smears demonstrate a slightly higher percentage of cell staining and stronger intensity of c-erbB-2 expression than the matched, air-dried smears. This is a sensitive and simple processing method that can be routinely applied in surgical pathology or fine needle aspiration biopsy specimens for the detection of c-erbB-2 (HER-2), with clinical implications.     

Acid alpha-naphthyl acetate esterase assay in murine lymphocytes

Acid alpha-naphthyl acetate esterase (ANAE) activity was assayed in cell homogenates and in intact cells by an endpoint colorimetric method, in which sodium dodecyl sulfate was used to stop the reaction. Each method of cell disruption and enzyme solubilization tested here caused a partial loss of the ANAE activity in lymphocyte preparations. The majority of the ANAE activity in lymphocytes was found to be membrane bound. The ANAE activity in thymocytes was over two times lower than that obtained for lymph node and spleen lymphocytes. Macrophages were found to contain about 18 times higher ANAE activity than mature lymphocytes.     

Increased numbers of alveolar macrophages with apoptotic bodies predict lung carcinoma

In our previous study on fixed tissue blocks, we reported a high apoptotic rate in patients with operated small cell lung carcinomas. In addition to tumour cells, numerous apoptotic bodies could also be found within alveolar macrophages within and close to tumour tissue. In order to test if such cells could be found in sputum smears and if their presence could be utilized as a marker in tumour diagnosis, we analyzed the occurrence of alveolar macrophages with apoptotic bodies (AMWABs) in a set of sputum smear and BAL samples from patients with and without a pulmonary malignancy. An increased amount of AMWABs in the cytoplasm could be found in sputum and BAL samples from patients with lung cancer. Interestingly, AMWABs could also be seen in patients with a histologically confirmed pulmonary malignancy, but with no detectable tumour cells in their sputum smear. Thus, the presence AMWABs in sputum smears could serve as a more sensitive marker of pulmonary malignancy than the prese nce of malignant cells per se. This is the first report describing apoptotic bodies in macrophages and the utility of their detection in cancer diagnosis.     

Identification of Campylobacter pylori in gastric biopsy smears

The use of gastric biopsy imprint smears to diagnose Campylobacter pylori was compared with the use of tissue sections and cultures. Multiple gastric biopsies were taken from the mucosa of 42 patients during endoscopy. Imprint smears were prepared from the samples used to make tissue sections; other samples were used for microbiologic culture. There was a good concordance (93%) between the morphologic diagnosis of C pylori in the air-dried, Giemsa-stained smears and the tissue sections; the cytologic preparations were clearly positive in six cases (14%) whose sections contained low numbers of the organisms. There was a concordance of 83% between the combined morphologic techniques and the bacteriologic culture. Six positive cases were detected only by the morphologic techniques while one positive case was detected only by bacteriologic culture. C pylori was identified in one or more preparations of the antral biopsy specimens in 23 (55%) of the 42 cases, including 23 (74%) of the 31 cases with a final diagnosis of gastritis or ulcer. These results show the usefulness of the cytologic study of gastric biopsy smears in diagnosing C pylori infections.     

Primary rhabdomyosarcoma of the prostate. Diagnosis by needle biopsy and immunocytochemistry

Needle aspiration biopsy of the prostate in a patient who initially presented with multiple metastatic lesions in the lungs and a pleural effusion showed the presence of many malignant cells. These cells appeared either as noncohesive round and oval cells or as clusters. The cytologic impression that these were sarcoma cells was confirmed by subsequent histologic and immunochemical studies. By correlating the cytologic, histologic and immunochemical findings with the clinical findings, it was possible to determine that the primary site of the tumor in this patient was the prostate. Our experience in this case suggests that air-dried smears stained with Romanowsky stains are suitable and useful for cytodiagnosis.     

Vaginal intraepithelial neoplasia and the Pap smear

OBJECTIVE: To assess the sensitivity of the vaginal smear cytologic examination in detecting vaginal intraepithelial neoplasia (VAIN) and to evaluate the cytologic findings of cases of VAIN. STUDY DESIGN: Cases with a histologic diagnosis of VAIN were identified from the Barnes-Jewish Hospital South and North Campus over a period of five and nine years, respectively. Only posthysterectomy patients with a tissue biopsy diagnosis of VAIN and with a vaginal smear obtained within three months of the biopsy were included in the study. Pertinent clinical information was obtained by reviewing the medical records. Two pathologists reviewed the pathologic samples. RESULTS: Thirty-five vaginal smears from 31 posthysterectomy patients were included in the study. The mean age was 57 years (range, 29-84). The cytologic diagnoses of smears from patients with VAIN included: high grade squamous intraepithelial lesion (19 cases), low grade squamous intraepithelial lesion (10 cases), atypical squamous cells of uncertain significance (5 cases) and negative for malignancy (1 case). CONCLUSION: The sensitivity of the vaginal smear cytologic examination in detecting VAIN is 83%. Obscuring inflammation contributed to false negative diagnoses in two cases.     

Inflammatory atypia and the false-negative smear in cervical intraepithelial neoplasia

The effectiveness of cervical cytologic screening is compromised by the increasingly recognized prevalence of false-negative smears. Our previous studies suggested that some false-negative cytologies can be accounted for by smears showing cervical intraepithelial neoplasia (CIN) reported as inflammatory atypia; we found that at least 4% of 5,752 consecutive smears had been underreported in this manner. In the present study, that data was reanalyzed to derive 95% confidence limits for the number of CIN smears reported as inflammatory atypia. Using several differing estimates of cytologic screening sensitivity, it is speculated that, under certain testable assumptions, colposcopy of patients with cytologic diagnoses of inflammatory atypia may be one cost-effective approach to finding CIN cases missed by screening. If confirmed, these findings imply that laboratory quality assurance efforts, traditionally directed to the most serious cytologic diagnoses, should also focus in part on nondysplastic atypia.