Pulmonary cytology in paracoccidioidomycosis

Paracoccidioidomycosis caused by the fungus Paracoccidioides brasiliensis is a common endemic deep mycosis in Brazil and other Latin American countries; the lungs are frequently involved with suppurative and granulomatous inflammation. With the aim of using pulmonary cytology as a diagnostic tool in paracoccidioidomycosis, the cytologic findings in 127 sputa, 4 bronchial washings and 2 bronchial aspirates from 45 patients with the mycosis were reviewed. Smears from all samples were stained by the Shorr and Leishmann techniques. Cell-block preparations stained with hematoxylin and eosin and by the Gomori-Grocott method were available from 115 samples. Most samples (55%) were purulent, 30% were hemorrhagic and 17% were mucous. Polymorphonuclear neutrophils, macrophages and multinucleated giant cells were observed in all cases. P. brasiliensis was identified in samples from 95.5% of the patients, more frequently in the cell-block preparations (93%) than in the smears (57.7%), probably as the consequence of the application of the Gomori-Grocott stain to the former. Epithelioid cells were present in 62.2% and squamous metaplasia of the respiratory epithelium in 51.1% of cases. Cytology of pulmonary samples proved to be a useful diagnostic method for the detection of lung involvement by paracoccidioidomycosis in humans. The accuracy of the method increased with the number of samples examined from each patient.     

Identification of Pneumocystis carinii in immunodeficient mice

Various procedures were utilized to determine the most sensitive, cost and labor effective techniques for detection of Pneumocystis carinii in immunologically compromised mice. Immunoperoxidase staining techniques that utilized polyclonal antibodies directed against purified rat or mouse P. carinii were more sensitive and specific than staining with Gomori's methenamine silver. Indirect immunofluorescence microscopy on frozen sections was comparable to immunoperoxidase staining, but lacked fine cytologic detail. Impression smears were of limited value when stained with Diff-Quik Stain, Harleco's Hemacolor, Wright-Giemsa or Wright-Leishman stains. However, cysts could be detected consistently in imprints stained with Gomori's methanamine silver. Transmission electron microscopy showed ultrastructural detail of P. carinii, but this technique was too costly and time consuming for routine use. Thus, because of its sensitivity and specificity, immunohistochemistry on paraffin sections was the most satisfactory method for screening and identifying P. carinii in lungs of immunocompromised mice.     

Extrapulmonary Pneumocystis carinii infection in an AIDS patient: a case report

BACKGROUND: Extrapulmonary Pneumocystis carinii (EPC) infection is an uncommon condition, regardless of HIV status, and can occur as a complication of P carinii pneumonia (PCP). However, PCP is the most common severe opportunistic infection in patients with AIDS. The incidence of EPC is variable, and in HIV-1-infected individuals it has been estimated to be 0.06-2.5%. CASE: A case of generalized lymphadenopathy was referred to us for fine needle aspiration cytology (FNAC). The patient was a 9-year-old boy who had a toxic facies and manifested multiple skin lesions all over the body. Fever was present during the examination. HIV status was confirmed from the history and test report. FNAC was done from a cervical lymph node and smears stained with hematoxylin-eosin and with Giemsa and Papanicolaou stain. The presence of P carinii was suspected in Giemsa- and hematoxylin-eosin-stained smears, and silver methenamine stain was used to confirm the diagnosis. Fungal spores were seen as small, spherical cysts of variable sizes, more or less the size of erythrocytes. The diagnosis was thus established as EPC infection. CONCLUSION: Lymph node involvement is the most common site of pneumocystosis in AIDS patients. Fine needle aspiration diagnosis of EPC infection is a possibility in such cases with lymphadenopathy and must be included in the differential diagnosis of lymph node swellings in AIDS.     

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).     

Cytodiagnosis of Campylobacter pylori in Papanicolaou-stained imprints of gastric biopsy specimens.

The use of Papanicolaou-stained touch preparations of gastric antral biopsies for the identification of Campylobacter pylori was examined using specimens obtained from 63 consecutive patients with endoscopic evidence of antral gastritis, with the results compared to routine histologic examination and Warthin-Starry silver staining. Organisms were readily identifiable in the Papanicolaou-stained imprints of the gastric mucus. The sensitivity in detecting organisms was 92.5% for the Warthin-Starry-stained sections, 71.4% for the Papanicolaou-stained imprints and 100% for both techniques combined. False-negative imprints were attributed to poor smears and/or the submission of duodenal tissue rather than antral biopsies. Properly performed touch preparations stained by the Papanicolaou method are a cost-effective adjunct to Warthin-Starry-stained section for improving the sensitivity of gastric biopsies for the diagnosis of C pylori.     

Ag-NOR technique in fine needle aspiration cytology of salivary gland masses.

Because of their complexity, salivary gland lesions are often difficult to identify correctly with fine needle aspiration cytology. To see whether the Ag-NOR staining technique for nucleolar organizer regions would be useful in this respect, we studied a series of smears from benign and malignant salivary gland lesions. The smears, previously treated with Papanicolaou and May-Grünwald-Giemsa stain, were destained and restained with Ag-NOR silver. The correlation between the cytologic-histologic diagnosis and the number of Ag-NORs in benign (sialadenitis, pleomorphic adenoma, oncocytoma and Warthin's tumor) and malignant lesions (adenoid cystic carcinoma, adenocarcinoma, carcinoma ex pleomorphic adenoma and squamous carcinoma) was statistically significant (P = less than .05). The Ag-NOR technique appears useful in the diagnosis of salivary gland lesions. One great advantage is that previously stained slides can be reused for silver staining, thus providing an excellent guide to the diagnosis, especially in doubtful cases and when corresponding histologic specimens or extra unstained slides are unavailable.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

AgNOR quantification in the diagnosis of follicular pattern thyroid lesions

OBJECTIVE: To evaluate the usefulness of silver staining of nucleolar organizer regions (AgNORs) in the preoperative diagnosis of follicular lesions in the thyroid with computer-aided morphometric analysis of the silver dots. STUDY DESIGN: Forty-eight cytologic smears of the thyroid were divided into 3 groups according to the results of postoperative histopathologic examination: hyperplastic nodules in nodular goiter (NG) (20), follicular thyroid adenoma (FTA) (20) and follicular thyroid carcinoma (FTC) (8). They were silver stained. The slides were analyzed with a computerized system for image analysis. Nearly 20 variables describing AgNORs were calculated (related to the area of the dots, number of dots and intranuclear localization of the dots). RESULTS: Only assessment of the total area of AgNORs in the nucleus allowed distinguishing between malignant and benign lesions. It was possible to determine the cutoff value of the total area of AgNORs in the nucleus (3.00 microns 2), limiting FTC from other lesions (observed ranges: NG, 1.64-2.87 microns 2; FTA, 1.81-2.85 microns 2; FTC, 3.01-3.97 microns 2). Evaluation of the mean number of AgNORs per nucleus did not improve the diagnosis of malignancy. CONCLUSION: Computer-aided morphometric analysis of silver dots may be useful in the preoperative diagnosis of thyroid lesions.     

The distribution of alpha 1-antichymotrypsin and amyloid production in the brain in Alzheimer's disease.

In this immunohistopathological study alpha 1-antichymotrypsin, which is barely demonstrable in the normal brain, was found in amyloid fibrils, endothelial cells and the cytoplasm of astroglial cells in brains from patients with Alzheimer's disease. Amyloid precursors stained with methenamine silver were arrayed mainly along the membranes, and amyloid fibrils, which stained densely with anti-alpha 1-antichymotrypsin, were in direct contact with the fibrous structures connecting with the membranes of vascular feet or astrocytic processes. From the above findings, alpha 1-antichymotrypsin seems to play a role in the production of amyloid fibrils in Alzheimer's disease.     

A triage system for processing fine needle aspiration cytology specimens

Fine needle aspiration (FNA) cytology is increasingly used in the operating room, in radiology suites and in physicians' offices. A major cause of nondiagnostic FNA specimens is poor preparation techniques. To avoid this problem, we have developed a triage system for handling and preparing FNA specimens. (1) For aspirations performed without a cytotechnologist present, the aspirate is rinsed into 50% ethanol; Cytospin slides are then prepared and stained by the Papanicolaou method. Clinicians are disposed toward this indirect method since no special slide preparation techniques are necessary. The results are equal to those of direct smear preparations, and material so prepared may also be used for immunocytochemistry. (2) When a cytotechnologist is present, both direct and indirect preparations are made. (3) When an immediate interpretation is desired, to ensure adequacy and/or to make a diagnosis, alcohol-fixed direct smears are stained with a modified toluidine blue-eosin stain and read by the cytopathologist. This offers the advantage of a quick diagnosis, which may be made in less than 90 seconds, but with nuclear detail equal to that of the Papanicolaou stain and with good background staining. In summary, our three-tiered approach offers optimal processing for aspirates collected in various settings in our institution.     

Small forms and hyphae of Paracoccidioides brasiliensis in human tissue

A 53 year-old man had a three-year recurrent respiratory infection. No fungi was detected in sputum examinations. Immunodiffusion test with paracoccidioidin revealed two precipitin bands. Very small forms and hyphae of a fungus were seen on silver methenamine stained serial sections from lung's lesion. P. brasiliensis was identified on the basis of the rare multibudding small forms.     

Hypercalcemia and disseminated histoplasmosis in an owl monkey

An adult male owl monkey (Aotus nancymai) had hypercalcemia associated with a disseminated case of histoplasmosis (Histoplasma capsulatum var. capsulatum). Histopathologic examination of tissues at necropsy showed moderate to marked numbers of small granulomas involving multiple organs. The granulomas contained numerous round, basophilic, intracellular structures, 1-2 microns in diameter, that stained positive in Periodic acid-Schiff and Gomori's methenamine silver tests. The hypercalcemia in this case was attributed to enhance conversion of dietary vitamin D to 1,25-dihydroxycholecalciferol by the granulomas.     

Modifications of Kohn's chlorazol black E staining and Wheatley's trichrome staining for temporary wet mount and permanent preparation of Entamoeba histolytica.

Preparation of stained smears of Entamoeba histolytica has several drawbacks. We therefore tried to simplify the staining procedures by modifing Kohn's chlorazol black E staining and Wheatley's trichrome staining techniques. Trophozoites and cysts of axenically cultured E. histolytica and Entamoeba invadens, respectively, and trophozoites and cysts of E. histolytica in stools of patients were used. Karyosomes and peripheral chromatin of nuclei and chromatoid bodies became distinctly visible after amoebae were suspended in the basic solution of Kohn's stain. Amoebae fixed in suspension with either basic solution or Bouin's fixative were clearly stained with Kohn's and trichrome preparations, both as wet mounts directly and as permanent slides after processing for mounting. These procedures were easier when the basic solution was used as a fixative and trichrome stain was employed. Erythrocytes ingested by trophozoites, however, were not stained with either of these preparations after fixation in the basic solution but were clearly stained when Bouin's fixative was used. Cysts of E. histolytica in stools concentrated using basic solution (instead of formalin) and ether were also stained with these stains. Consequently, without employing highly toxic mercuric chloride, wet mounts and permanent smears can be prepared with permanent stains, and preserved cysts can be stained after concentration.     

Pneumocystis Carinii Pneumonia: Relationship Between Cyst Size and Response to Treatment

Pneumocystis carinii pneumonia is a common cause of death in patients with AIDS. Diagnosis is based on cytological examination of smears prepared from induced sputum samples and bronchoalveolar lavage (BAL) specimens which have been stained with methenamine silver. We have examined 46 BAL/induced sputum specimens from patients who had clinical symptoms and signs suggestive of P. carinii pneumonia and measured the diameter of a minimum 100 cysts in each specimen. We found that cyst size correlated with response to treatment with co-trimoxazole. This observation has implications for the therapeutic management of patients with this infection. La pneumopathie áPneumocystis carinii est une cause de décès fréquente chez les patients atteints d'un syndrome d'immunodéficience acquise (SIDA). Le diagnostic est basé sur l'examen cytologique des frottis préparés à partir du produit d'expectoration provoquée ou du liquide de lavage bronchoalvéolaire (LBA), après coloration de Grocott. Le diamètre d'au moins 100 kystes par échantillon a été mesuré dans 46 prélèvements de LBA/expectoration provoquée de patients présentant des signes cliniques suggérant une pneumopathie àPneumocystis carinii. Nous avons constaté que le diamètre des kystes est corrélé avec la réponse au traitment par co-trimoxazole, Cette observation a des implications sur la conduite du traitement des patients porteurs de cette infection. PCP ist eine häufige Todesursache bei HIV-Patienten. Die diagnose beruht auf dem cytologischen Nachweis in induzierten Sputumausstrichen oder bronchio-lo-alveolären Lavagen mit der Grokott-Färbung. In 46 positiven Fällen wurde der Durchmesser von mindestens 100 Cysten in jeder Probe gemessen und eine Korrelation zum Behandlungserfolg mit Co-Trimoxazol nachgewiesen.     

The distribution of α1-antichymotrypsin and amyloid production in the brain in Alzheimer’s disease

In this immunohistopathological study α₁-antichymotrypsin, which is barely demonstrable in the normal brain, was found in amyloid fibrils, endothelial cells and the cytoplasm of astroglial cells in brains from patients with Alzheimer’s disease. Amyloid precursors stained with methenamine silver were arrayed mainly along the membranes, and amyloid fibrils, which stained densely with anti-α₁-antichymotrypsin, were in direct contact with the fibrous structures connecting with the membranes of vascular feet or astrocytic processes. From the above findings, α₁-antichymotrypsin seems to play a role in the production of amyloid fibrils in Alzheimer’s disease.     

Examination of Induced Sputum In the Diagnosis of Pneumocystis Carinii Pneumonia

The results of the examination of sputum induced by the inhalation of nebulized hypertonic saline in the diagnosis of Pneumocystis carinii pneumonia (PCP) are presented. In suspected cases of PCP in patients who were either HIV antibody positive or were receiving immunosuppressive therapy, 46 induced sputum specimens were stained using both Grocott's modified Gomori methenamine silver nitrate (GMS) and immunofluorescence staining. In 12 specimens P. carinii cysts were detected by both methods, in four specimens by GMS staining only and in five specimens by immunofluorescence only. The sensitivity of induced sputum examination in the detection of P. carinii cysts was increased by using both of these staining methods on each sputum specimen and the need for more invasive methods of diagnosis was reduced.     

Oral exfoliative cytology in the diagnosis of paracoccidioidomycosis

OBJECTIVE: To assess the efficacy of conventional oral exfoliative cytology as a diagnostic tool in paracoccidioidomycosis. STUDY DESIGN: Cytologic smears and incisional biopsies were obtained from 10 patients with a clinical suspicion and oral manifestations of paracoccidioidomycosis. Cytologic smears and sections of the incisional biopsy underwent methenamine silver staining for fungi according to the Gomori-Grocott method. The dry glass slides were examined at 400 or 1,000 x magnification, and the presence and shape of yeasts of Paracoccidioides brasiliensis were investigated. RESULTS: Yeasts of the fungus P brasiliensis were clearly identified in cytologic smears and sections from incisional biopsies in all cases analyzed (100.0%). CONCLUSION: Cytology of oral samples proved an effective diagnostic method for the detection of paracoccidioidomycosis in humans.     

Scanning Feulgen-deoxyribonucleic acid cytophotometry of Papanicolaou destained preparations.

Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou destained specimens revealed a differential loss in Feulgen reactivity among human buccal and cervical smears, cultured embryonic lung fibroblasts and invasive cervical carcinoma cells. Loss in Feulgen reactivity in Papanicolaou destained fibroblasts and polyploid nuclei of malignant lesions was observed to result in underestimates of relative Feulgen deoxyribonucleic acid and nuclear area values using scanning integrating microdensitometry. Thus, Papanicolaou stained preparations may not be suitable for deoxyribonucleic acid quantification of high ploidy lesions since distributional absorption error is unpredictably influenced by such factors as ploidy level, nuclear size, chromatin dispersion and differential aldehyde loss during destaining. Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou stained preparations can be useful for differentiating benign from malignant lesions if extent of aneuploidy (as reflected in abnormal deoxyribonucleic acid frequency distribution profile) is used as a diagnostic indicator.     

Direct Fluorescent-Antibody Method for the Diagnosis of Pneumocystis carinii Pneumonitis from Sputa or Tracheal Aspirates from Humans

A direct fluorescent antibody (DFA) method was applied to sputum or tracheal aspirate from 68 patients with clinical or radiological evidence suggesting Pneumocystis carinii pneumonitis, and to 50 control patients. P. carinii was detected by DFA in specimens from 33 of the 69 clinical cases and 3 of the 50 controls. Specimens of lung from 11 of 33 DFA-positive cases were examined histologically, and 9 were positive. Four of 35 DFA-negative cases were examined histologically, and all were negative. Sputa or tracheal aspirates from 6 patients who were positive by both DFA and histological examination were examined also by methenamine silver staining; none could be diagnosed conclusively by this method. The results indicate that the DFA method is a sensitive and dependable procedure for the laboratory diagnosis of P. carinii pneumonitis in man.     

Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours

U.S. Choi and D.Y. Kim Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours Objective: To investigate whether Diff‐Quik stained fine needle aspirate smears can be used to evaluate Ki‐67 expression by immunocytochemistry. Methods: Both cytological and histological samples were obtained from 24 dogs with spontaneously developed mammary gland tumours. The cytological and histological specimens were examined by Diff‐Quik and H&E stains, respectively. After examination, both samples were immunostained using the same Ki‐67 antibody. The % Ki‐67 values were calculated based on the percentage of positively stained tumour cells per 500 and 1000 tumour cells in cytology and histology specimens, respectively. Results: Ki‐67 staining was successful in 17/24 smears (71%) and 19/23 sections (83%). The correlation coefficient between the percentage of Ki‐67‐positive cells in cytological smears and in the histological sections was 0.677 (P < 0.01). These values were significantly different between histologically benign and malignant tumour groups both in cytology and histology samples (P < 0.001). The threshold value of the percentage of Ki‐67‐positive cells for distinguishing benign from malignant tumours was set at 4.85% with 90.9% sensitivity and 92.3% specificity by Receiver Operating Characteristic (ROC) curve using histopathology as the gold standard. Conclusion: Diff‐Quik‐stained cytology smears can be used to detect the presence of Ki‐67 antigen when histology sections are not available.