Comparison of formaldehyde and methanol fixatives used in the detection of ion channel proteins in isolated rat ventricular myocytes by immunofluorescence labelling and confocal microscopy.

In this study, a fixation protocol using a 10% neutral buffered formalin (FA) solution and another protocol using a methanol (MeOH) solution were compared for detection of ion channels, Kv1.5, Kv4.2, Cav1.2, Kir6.2, Nav1.5 and Nav1.1 in rat myocytes by immunolabelling. Kv1.5 and Kv4.2 at intercalated discs and Cav1.2 at transverse tubules were not detected by FA but were detected by MeOH. Kir6.2 at transverse tubules and Nav1.5 at sarcolemma were detected by FA but not by MeOH. It is suggested that both FA and MeOH fixation protocols should be used for the detection of cardiac ion channels by immunolabelling.

Diagnostic value of urine erythrocyte morphology in the detection of glomerular disease in SurePath™ liquid-based cytology compared with fresh urine sediment examination.

OBJECTIVE: To assess whether the morphology of urine erythrocytes can be an effective tool for distinguishing glomerular disease from lower urinary tract disease in SurePath™ liquid-based cytology (SP-LBC). METHODS: We examined four morphological parameters of erythrocytes: (1) irregular erythrocytes (of all types including fragmented forms) comprising greater than or equal to 20% of erythrocytes; (2) uniform erythrocytes (>80%); (3) doughnut or target-like shaped (D/T) erythrocytes (≥1%); and (4) acanthocytes (≥1%) in glomerular disease (n = 32) and lower urinary tract disease (n = 20) with SP-LBC slides in cases that had also been assessed by fresh urine sediment examination. RESULTS: Sensitivity of D/T erythrocytes and acanthocytes (dysmorphic erythrocytes) for glomerular disease were 100% and 87.5%, respectively, with urine sediment examination, and 81.3% and 46.9%, respectively, in SP-LBC slides. Specificity was 100% for D/T erythrocytes and acanthocytes using either procedure. While irregular erythrocytes were specific for glomerular disease using urine sediment examination, they were seen in 70% of those with lower urinary tract disease using SP-LBC slides as a result of the deformation of erythrocytes by the fixative. CONCLUSIONS: Although the sensitivity of D/T erythrocytes and acanthocytes for glomerular disease was lower in SP-LBC slides than fresh urine sediment examination, their specificity was equally high. Therefore, urine erythrocyte morphology is useful in the detection of glomerular disease with the SP-LBC slides. However, morphological features apart from D/T erythrocytes and acanthocytes are not useful in SP-LBC slides.

Expression of vimentin and high-molecular-weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low-grade urothelial carcinoma cells in voided urine.

OBJECTIVE: Reactive renal tubular cells show features of an atypical repair reaction. Differentiation between reactive renal tubular cells and low-grade urothelial carcinoma (LG-UC) cells can therefore be a diagnostic challenge based on morphology alone. In this study, we evaluated the diagnostic utility of vimentin and a high-molecular-weight cytokeratin antibody (clone 34ßE12) in differentiating reactive renal tubular cells from LG-UC. METHODS: We evaluated voided urine cytology and surgical specimens from 40 patients with renal disease, and 17 patients with LG-UC. All slides were stained with vimentin and 34ßE12. RESULTS: In the reactive renal tubular cells in voided urine cytology, vimentin showed strong cytoplasmic staining in 39/40 (97.5%) cases, but all were negative for 34ßE12. LG-UC cells showed positive staining for 34ßE12 in 3/17 (17.6%) cases, whereas none were positivity for vimentin. The reactive renal tubular cells of histological specimens in the renal disease group demonstrated positive for vimentin in all 40 cases and all were negative for 34ßE12. The LG-UC group showed abnormal staining for 34ßE12 in 4/17 (23.5%) cases, whereas none were positive for vimentin. CONCLUSIONS: Vimentin expression in urine cytology can help to distinguish reactive renal tubular cells from LG-UC. However, 34ßE12 does not appear to be a useful adjunct to distinguish these two groups in voided urine cytology.

Value of computer-assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low-grade urothelial carcinoma.

OBJECTIVE: To assess whether computer-assisted quantitative morphological parameters can be an effective tool for objectively distinguishing reactive renal tubular cells from low-grade urothelial carcinoma cells (LG-UCs) in voided urine. METHODS: Nuclear morphometry was performed by a computer-assisted image analyser system on Papanicolaou-stained cytological specimens. The circumference of reactive renal tubular cells (n = 40) or LG-UC (n = 20) nuclei were manually traced, and the following nuclear morphometric parameters were analysed: (i) area, (ii) perimeter, (iii) roundness factor, (iv) maximum length, and (v) linear factor. For each nuclear measurement, we calculated the maximum, minimum, mean and standard deviation. RESULTS: The mean nuclear area and nuclear perimeter were higher in reactive renal tubular cells compared to the LG-UCs. The mean of roundness and linear factors (reflecting a tendency for the nuclear outline to be regular and oval, respectively) were higher in LG-UCs compared with reactive renal tubular cells. Among nuclear areas, the nuclear perimeter, roundness factors and maximum length did not show any significant differences between reactive renal tubular cells and LG-UCs. On the other hand, the linear factor showed a mean higher value among LG-UCs than reactive renal tubular cells (P = 0.023). CONCLUSIONS: Of five quantitative nuclear morphological parameters, only linear factor was statistically significant in differentiating reactive renal tubular cells in renal disease from LG-UCs.

Can cytological features differentiate reactive renal tubular cells from low-grade urothelial carcinoma cells?

OBJECTIVE: To compare the cytomorphological and immunocytochemical features of reactive renal tubular cells and low-grade urothelial carcinoma cells (LG-UCs). METHODS: We examined 15 cytological parameters in 38 cases with reactive renal tubular cells in renal disease and 20 cases of LG-UCs from bladder cancer that had been diagnosed by histological examination. Voided urine cytological parameters evaluated were as follows: (i) maximum cell numbers of clusters, (ii) cannibalism, (iii) rosette-like arrangement, (iv) hobnail-shaped cells, (v) vacuolated cytoplasm, (vi) intracytoplasmic haemosiderin, (vii) irregular nuclear contours, (viii) chromatin pattern, (ix) prominent nucleoli, (x) cast encasement, (xi) casts, (xii) dysmorphic erythrocytes, (xiii) isomorphic erythrocytes, (xiv) necrosis, and (xv) vimentin reactivity. The above parameters were determined using Mann-Whitney U-test and chi-square test, with differences considered significant at P < 0.05. RESULTS: In reactive renal tubular cells, low to moderate cell numbers of clusters (fewer than 50 cells), rosette-like arrangement, hobnail-shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, euchromatin pattern, prominent nucleoli, dysmorphic erythrocytes and vimentin reactivity were present in significantly higher proportions compared with those in LG-UCs. In LG-UCs, high cell numbers of clusters (50 cells or more), cannibalism, heterochromatin pattern, isomorphic erythrocytes and necrosis were seen in significantly higher proportions. No significant differences were observed in irregular nuclear contours, cast encasement or casts. CONCLUSIONS: Based on results of the present study, maximum cell numbers of clusters, cannibalism, rosette-like arrangement, hobnail-shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, chromatin pattern, prominent nucleoli, dysmorphic erythrocytes, isomorphic erythrocytes, necrosis, and vimentin reactivity were capable of distinguishing reactive renal tubular cells from LG-UCs.

'Cannibalism' (cell phagocytosis) does not differentiate reactive renal tubular cells from urothelial carcinoma cells.

OBJECTIVE: Cannibalism of one cell by another in voided urine cytology has been considered a cytological feature for differentiating urothelial carcinoma (UC) from benign lesions. Recently, however, we observed cannibalism in voided urine obtained from patients with renal glomerular disease (RGD). The purpose of this study was to determine the cytomorphological and immunocytochemical characteristics of cannibalism in voided urine from RGD. METHODS: Seventy cytology specimens of voided urine were examined and the findings were compared with the histological findings. In addition, we compared the cytomorphological and immunocytochemical differences in cannibalism found in RGD and cases of UC selected as showing cannabilism. RESULTS: Cannibalism in voided urine was found in three (5.5%) of 55 RGD cases. The finding was measured as (1+) < 5 cells, (2+) 5-20 cells, and (3+) > 20 cells and was (1+) in all three RGD cases, compared with 6.7%, 60% and 33.3% respectively in 15 UC cases. Differences in low cellularity cases (1+) and moderate to high cellularity cases (2+ or 3+) were statistically significant between RGD (3 and 0) and UC (1 and 14) (P=0.005). The maximum diameter of cannibalized cells in RGD was 24.3-33.0 microm (mean 29.8 microm) versus 18.0-30.4 microm (mean 23.3 microm) in UC (P=0.004). Necrosis and isomorphic erythrocytes were absent in RGD, but were found in 46.7% and 86.7%, respectively, of UC cases (P=0.245 and P=0.012). Dysmorphic erythrocytes were identified in all three cases with RGD and 13.3% of UC (P=0.012). Vimentin reactivity was found in all cases with cannibalism in RGD, but never in UC (P=0.001). CONCLUSIONS: Our results demonstrated that cannibalism in voided urine is present not only in UC but also in RGD. Furthermore, we showed that cellularity of cannibalism, vimentin reactivity and background differed significantly and can be used for differential diagnosis between the two groups.

A case of infantile rhabdomyofibrosarcoma with immunohistochemical, electronmicroscopical, and genetic analyses.

A case of infantile rhabdomyofibrosarcoma arising on the buttocks of a 15-month-old boy is reported with histological, immunohistochemical, electronmicroscopical, and cytogenetic findings. Histological examination showed a proliferation of spindle-shaped cells in a fasciculated pattern, with occasional rounded rhabdomyoblastic cells with abundant eosinophilic cytoplasm. Immunohistochemically, the tumor cells expressed desmin and MyoD1 but were only weakly positive for myoglobin. No clear rhabdomyoblastic features were observed by electronmicroscopic examination. Chromosome analysis showed a clone of 46, XY, der(2)t(2;11)(q37;q13), different from any karyotypic abnormality in the original report of this neoplasm. Loss of heterozygosity at 11p15.5, the most frequent genetic alteration in embryonal rhabdomyosarcoma, was not detected. The low degree of striated muscle differentiation and tumor localization supported the diagnosis of infantile rhabdomyofibrosarcoma rather than spindle-cell rhabdomyosarcoma in this case. The present case has been uneventful as of 25 months after surgery. The rather long recurrence-free period, which has not been reported in previous cases, may be attributable to chemotherapy-induced rhabdoid differentiation of the tumor cells.

Melanin pigmented oncocytic metaplasia of the nasopharynx.

A 64-year-old man presented with a history of discomfort of the throat of a few weeks' duration. Nasoscopic examination revealed multiple small, brown pigmentations at the left suprapharynx, the base of the left nasal cavity and the pharyngeal openings of the auditory tube on both sides. Microscopically, the lesion showed a glandular pattern of oncocytic epithelium with abundant pigmented granules and melanophages in the surrounding stroma. Immunohistochemically, the dendritic cells in the basal layer were positive for S-100 protein. Electron microscopic study revealed numerous fully melanized melanosomes and hypertrophied mitochondria in the oncocytic cells. Oncocytic cells do not produce melanin for themselves, melanin granules apparently being transferred from the adjacent dendritic cells to the oncocytic cells.

Collision tumor of cutaneous malignant melanoma and basal cell carcinoma.

An extremely rare case of a collision tumor of malignant melanoma (MM) and basal cell carcinoma (BCC) located in the temporal scalp of a 78-year-old male is reported. Microscopic examination revealed a collision tumor consisting of the nodular type, Clark's level IV MM, and a pigmented BCC. The tumor was studied by immunohistochemistry and was analyzed for point mutations of codons 12 and 61 of H-ras, K-ras and N-ras oncogenes. Tumor cells of the MM were positive for S-100 protein, HMB-45 and vimentin, but negative in the BCC. The malignant melanoma possessed an activating A to T transversion in codon 61 of N-ras gene, which is the code for leusine instead of glutamine. This is the first report of ras oncogene mutation in such a collision tumor. MM and BCC are clearly different in terms of histological, immunohistochemical and ras gene mutation analyses. These findings support the view that the association of MM and BCC in this collision tumor is a chance occurrence.

Ascitic fluid cytology of adenosarcoma of the ovary: a case report.

Extrauterine adenosarcoma is very rare and originates in the ovary, adnexa, or myometrium. Cytologic study of ascites is very important to determine clinical staging of malignant ovarian tumors and provide adequate therapy for recurrence. The cytomorphologic features of adenosarcoma have been only rarely described. A 77-yr-old woman visited a hospital with a complaint of lower abdominal pain for 1 mo. A tumor originating from the right adnexa in the pelvis, and involving the rectum, was found in surgery. In the ascitic fluid cytology, a few dispersed tumor cells with large cytoplasm and nuclei were oval-shaped, with nuclear invagination. The chromatin was finely granular; one or two nucleoli were conspicuous. To our knowledge, this is the fifteenth reported case of adenosarcoma of the ovary, and there have been no prior reports describing the cytological features of ascitic fluid cells in adenosarcoma of the ovary.

Squash cytology of pleomorphic xanthoastrocytoma mimicking glioblastoma. A case report.

BACKGROUND: Pleomorphic xanthoastrocytoma (PXA) is an uncommon, superficially located and well-circumscribed brain tumor that originates in astrocytic cells. Despite the fact that the tumor cells are pleomorphic, with bizarre nuclei, the clinical course is favorable. Cytologic and histologic differentiation from other high grade gliomas is necessary to determine adequate therapy during surgery. Cytomorphologic features of this tumor have been described only rarely. CASE: A 22-year-old male had complained of visual disturbance for about a year. Radiologic imaging revealed a well-circumscribed mass with cyst formation in the left temporal area. Squash specimens from fresh tissues were highly cellular. Tumor cells were markedly pleomorphic, with long and coarse cytoplasmic processes showing a fibrillary astrocytic appearance. Pleomorphic cells varied in shape from round to elongated and had large, multilobed, hyperchromatic nuclei but few nuclear mitoses. Sometimes eosinophilic granular bodies were also observed. Blood vessels were found frequently in tumor cell clusters, but their endothelium was not swollen. In the background, considerable leukocytic infiltration, but no cellular debris, was observed. With immunohistochemical studies, most of the tumor cells were positive for glial fibrillary acidic protein and S-100 protein. Some of the mononuclear giant cells were positive for synaptophysin. CONCLUSION: Squash preparations showed the peculiar cytologic features of PXA. Together with the peculiar radiologic findings, the cytologic results make it possible to render a diagnosis of PXA.

Meningeal rhabdomyosarcoma. Report of a case with cytologic, immunohistologic and ultrastructural studies.

Primary rhabdomyosarcoma of the meninges, a very rare brain tumor, is reported. Cytologic findings by squash preparation were useful as an adjunct to frozen section diagnosis during surgery. The cytologic features of rhabdomyosarcoma without cross-striation have some similarities to those of gemistocytic astrocytomas and anaplastic meningiomas, but the cytoplasmic filaments of rhabdomyosarcoma are different from those of gemistocytic astrocytoma and anaplastic meningioma. Histologically the tumor was embryonal rhabdomyosarcoma with a partially botryoid pattern. The tumor cells have no cross-striations but react positively to antimyoglobin serum on immunoperoxidase staining. In this case, intracytoplasmic filaments resembling poorly formed myofibrils were found on electron microscopic study. The histologic and immunohistologic findings suggest that this rare mesenchymal malignancy might arise from primitive totipotential cells.

Cytology and immunohistochemistry of anaplastic meningiomas in squash preparations. A report of two cases.

Cytodiagnosis is a useful adjunct to frozen section diagnosis of brain tumors during surgery and has been applied in many pathology laboratories. Although anaplastic (malignant) meningiomas are not extremely rare tumors, their cytologic features have not been well described. Cytologic details of anaplastic meningioma, papillary meningioma with a fibroblastic component and anaplastic angiomatous meningioma are presented. Papillary meningioma showed atypical cell clusters having a close relation to mature endothelial cells and that corresponded to papillary proliferation. Other cell clusters of this tumor showed an ordinary fibroblastic pattern with coarse cytoplasmic filaments and necrotic changes in the cytoplasm. Anaplastic angiomatous meningioma exhibited loose cell clusters. Some of them had coarse cytoplasmic filaments, and others were similar to epithelial cells in origin. Specific features commonly found in anaplastic meningioma were hyperchromatic and pleomorphic nuclei and cytoplasmic filaments more obscure than those of the benign counterpart. Immunocytochemically the tumor cells in the papillary area of papillary meningioma had a positive reaction to antiserum of vimentin, cytokeratin and S-100 protein. The tumor cells of anaplastic plastic angiomatous meningioma showed intracytoplasmic positivity to antivimentin and anti-epithelial membrane antigen serum.

Three cases of extrapulmonary small cell carcinoma occurring in the prostate, stomach, and pancreas.

Small cell carcinomas are fairly common neoplasms in the lung, but tumors featuring similar histological profiles may occur in extrapulmonary organs. Three cases of small cell carcinomas occurring in the prostate (case 1), stomach (case 2), and pancreas (case 3) are presented. Production of hormones was demonstrated immunohistochemically in all cases. In case 2 alpha-fetoprotein (AFP) was elevated in the serum and observed immunohistochemically in tumor cells. Production of AFP is a distinctive feature, which has not been reported in the pulmonary and extrapulmonary cases of small cell carcinoma. Amplification and/or expression of myc gene family have been suggested to be related to the prognosis of pulmonary small cell carcinoma. Amplification of myc genes was not detected in any of our cases, but c-myc protein was demonstrated immunohistochemically in tumor cells of case 1.

[A case of urachal xanthogranuloma causing recurrent intestinal obstruction].

A 68-year-old male was admitted to our hospital with the chief complaints of lower abdominal pain and fever. There was a tender mass palpable in the lower abdomen. Plain abdominal X-ray film revealed multiple air-fluid levels with dilated small bowel loops, suggesting intestinal obstruction. Abdominal ultrasonography, computed tomography and magnetic resonance imaging revealed a solid mass extending from umbilicus to the bladder dome beneath the rectal muscle. There was normal mucosa of the bladder by cystoscopic examination. A urachal tumor was clinically suspected and en bloc removal of the mass, the remaining urachus, umbilicus, omentum and bladder dome was performed. The histological diagnosis was urachal xanthogranuloma. The patient has remained in good health without any recurrence for 6 months since the surgery. We discuss urachal xanthogranuloma in the literature.

Heat stress induces a glycosylation of membrane sterol in myxoamoebae of a true slime mold, Physarum polycephalum.

To know the very early events occurring after heat shock, the changes of membrane lipids were examined. Heat stress induced the production of a certain glycolipid in the myxoamoebae of Physarum polycephalum in a few minutes. The purified glycolipid was determined to be a poriferasterol monoglucoside by structural studies that was previously reported to be expressed during the differentiation of Physarum cells from haploid myxoamoebae to diploid plasmodia (Murakami-Murofushi, K., Nakamura, K., Ohta, J., Suzuki, M., Suzuki, A., Murofushi, H., and Yokota, T. (1987) J. Biol. Chem. 262, 16719-16723). The activity of UDP-glucose:poriferasterol glucosyltransferase (Murakami-Murofushi, K., and Ohta, J. (1989) Biochim. Biophys. Acta 992, 412-415) was also expressed rapidly after heat shock. Thus, the activation of sterol glucosyltransferase and the production of sterol-glucoside were considered to be important events that were involved in the signal transduction system to induce some succeeding heat-shock responses, such as the synthesis of heat-shock proteins.

Environmental magnetic fields change complementary DNA synthesis in cell-free systems.

A number of studies have shown that exposures to environmental magnetic fields (MFs) increase cellular transcription and enhance DNA synthesis. However, little is known about the basic mechanisms underlying specific biological responses to MFs. We looked directly at the effect of MFs by using a cell-free rabbit globin cDNA synthesis system. cDNA synthesis reaction mixtures were placed in each of four exposure conditions and were tested simultaneously. Condition one was the control, with no exposure during either single-strand or double-strand synthesis (M-M-). The second condition was MF exposure only during single-strand synthesis (M+M-). The third condition was exposure only during double-strand synthesis (M-M+). The fourth condition was exposure during both single-strand and double-strand synthesis (M+M+). Results showed that cDNA synthesis was affected by 5-100 microT, 60 Hz MFs. Double-strand cDNA synthesis increased with MF exposure only during double-strand synthesis reactions (M-M+), and the greatest increase of double-strand cDNA synthesis was detected when MF exposure was at 10 microT, only during double-strand synthesis. Double-strand cDNA synthesis decreased when only single-strand synthesis reactions were exposed (M+M-; 100 microT). An increase of cDNA synthesis caused increased synthesis of rabbit globin cDNA and large-sized molecules. These results suggest that exposure to MF induced structural changes of synthesized cDNA, therefore altering the amount of cDNA. Our results show that environmental MFs can significantly alter cDNA synthesis in a cell-free system.

Processive movement of single 22S dynein molecules occurs only at low ATP concentrations.

We have analyzed the movement of single 22S dynein molecules from Tetrahymena cilia by using a nanometer measuring system equipped with optical tweezers. Statistical analysis proved that a single molecule of 22S dynein can move processively and develop force at low concentrations of ATP (<20 microM). The maximum force was approximately 4.7 pN, and the force-velocity curve was convex down. During force development, dynein molecules showed stepwise displacement of approximately 8 nm and frequently exhibited backward steps of approximately 8 nm. At higher concentrations of ATP (>/=20 microM) single molecules of 22S dynein were not observed to move processively. Twenty-two S dynein seems to switch over from a processive mode to a nonprocessive mode, sensing a subtle change of ATP concentrations. These observations indicate that the processivity, maximum force, and step size of dynein are similar to those of kinesin, but the ATP concentration-dependence, force-velocity relationship, and backward steps are clearly distinct from kinesin.

Effects of environmental level magnetic field exposures on transcription of CMV immediate early promoter DNA in a cell-free in vitro transcription system.

Effects of environmental levels of magnetic fields (MFs) on RNA synthesis have been investigated by using a cell-free system for in vitro transcription. Transcription reaction mixtures containing CMV immediate early promoter DNA plus HeLa cell nuclear extracts were exposed to each of three different MF field strengths, i.e., 10, 50, and 100 microT. Each MF exposed extract was paired with a simultaneous sham-exposure control. The present results show no significant differences in amounts of RNA synthesis in extracts of MF exposed compared with that in the sham controls. This finding is in contrast to results of prior studies of DNA synthesis in cell-free systems that showed MF exposure effects. The results of the present cell-free system studies suggest that the marked differences of MF exposure effects on DNA and on RNA synthesis direct attention to the complexity involved in confirming significant effects of exposures to environmental levels of MFs in biosystems in vivo and in vitro.