A sensitive, direct colorimetric assay of serum iron using the chromogen, nitro-PAPS.

A direct colorimetric method is presented for the determination of serum iron in 0.1-ml sized samples, using a new, water-soluble, reagent, 2-(5-Nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol Na salt (epsilon 585 nm = 9.4 X 10(4) l/mol per cm). Interference of copper and zinc in sera can be eliminated entirely by forming copper- and zinc-thioglycollate complexes immediately upon the dissociation of the protein-bound iron, copper and zinc by thioglycollate and sodium dodecyl sulfate. The serum blank was minimized by the use of sodium dodecyl sulfate as a protein denaturant. Within-run and between-run precision (CV) were in the range of 0.7-2.9% and 1.1-3.6%, respectively, depending on the serum iron content. A good correlation (r = 0.995) was obtained between this method and the reference method proposed by the International Committee for Standardization in Hematology.

A highly sensitive colorimetric determination of serum zinc using water-soluble pyridylazo dye.

A colorimetric method for precise and accurate determination of zinc in serum is presented. Only 0.3 ml of sample is necessary, because of the use of a new, highly sensitive reagent, 2-(5-bromo-2-pyridylazo)-5-(N-n-propyl-N-3-sulfopropylamino)-phenol (epsilon 554nm = 1.3 X 10(5) 1 . mol-1 . cm-1), which is water-soluble and has long-term stability. Interference of iron and copper in serum can be removed by co-precipitation of the iron fluoride complex with trichloroacetic acid precipitated proteins and the copper dithiocarboxy sarcosine complex, respectively. Within-run and day-to-day precision (CV) are in the range of 0.3-3.5% and 1.9-3.1%, respectively, depending on the serum zinc content. A good correlation (r = 0.98, p less than 0.05) was obtained between this method and atomic absorption spectrometry. In contrast to previous colorimetric methods, the present method does not involve heating, extraction with organic solvents, or a cyanide masking system.

Usefulness of estrogen receptor detection using archival Papanicolaou-stained smears.

OBJECTIVE: To examine estrogen receptor (ER) detection using cytologic specimens and to compare the results with those obtained by the dextran-coated charcoal (DCC) method and enzyme immunoassay (EIA). STUDY DESIGN: Immunocytochemical staining was conducted on 60 cases of breast cancer resected at our hospital between April 1993 and November 1997 in which ER had been measured by DCC or EIA. Specimens for immunocytochemical staining were prepared by a cell transfer method using archival Papanicolaou-stained imprint smears, and ER staining was performed by the labeled streptavidin method using an anti-ER monoclonal antibody. These results were compared with those obtained by DCC or EIA. RESULTS: In immunocytochemical staining for ER, positive staining was observed in the nuclei of tumor cells. A good correlation was obtained between the immunocytochemical staining results and biochemical results. Five cases were positive in anti-ER staining but negative in biochemical tests, and two cases were negative in anti-ER staining and positive in biochemical tests. CONCLUSION: Unlike biochemical assays, the immunocytochemical method does not necessitate use of fresh frozen materials and can be performed even using archival Papanicolaou-stained smears. Immunocytochemical study is a highly useful method for routine ER determination.

Cytodiagnosis of malignant melanoma of soft tissue: report of a case with diagnosis by intraoperative cytology.

BACKGROUND: Malignant melanoma of soft tissue (MMST) is a rare tumor and consists of < 1% of all soft tissue neoplasms. There are few reports on its cytodiagnosis. CASE: A 14-year-old male attended the Department of Orthopedics, Juntendo University Urayasu Hospital, in August 1994 because of a painless tumor in the distal portion of the left thigh. Intraoperative imprint smear examination led to a diagnosis of malignant melanoma, and wide resection of the tumor, including the surrounding normal tissue, was performed. On cytologic examination, the background was relatively clean, with tumor cells distributing individually or in clusters. Under high magnification, the tumor cells were seen to contain a slightly enlarged, conspicuous nucleolus and large cell body. The cells varied in shape from polygonal to spindle shaped, with a few multinuclear giant cells. Melanin and glycogen were observed in varying degrees in the tumor cells. CONCLUSION: MMST can be diagnosed easily if melanin is observed in the cytoplasm. Even in the absence of melanin, the tumor has relatively characteristic cytomorphology. Intraoperative cytology is useful for an accurate diagnosis of the tumor.

[Relation between human papilloma virus DNA and expressions of p53 and p21 proteins in cervical lesions].

Oncogenic types of human papilloma virus (HPV) are known to be closely associated with cervical carcinoma. On the other hand, the oncogenic process is associated with various abnormalities in the mechanisms of cellular regulation. In this study, we detected the expressions of p53 and p21 proteins in cervical lesions by immunohistochemical techniques, and examined the relationship with HPV infection as well as the clinical usefulness of the results. Cervical biopsy specimens from 107 cases of cervical lesions were studied. HPV-DNA was detected by the hybrid capture method using probe A for low oncogenic types and probe B for high oncogenic types. Anti p21, anti-p53 antibodies were used to perform immunostaining. Point mutation in the p53 gene was analyzed by the DGGE method. High oncogenic HPV types were detected at high frequencies in CIN and SCC. In lesions associated with high oncogenic HPV, p53 protein was detected in 33.4% of the lesions and p21 protein in 36.3%. The p53 gene was analyzed in all cases, and point mutation was not detected. No relation was detected between HPV infection and p53/p21 protein expression. Since mutation was not found in the p53 gene, the p53 protein expressed was considered to be wild-type, which is suspected to play a role in inhibiting disease progression in some cases.

[Detection of HPV-DNA in the various uterocervical lesion by the in situ polymerase chain reaction].

The causal association of human papilloma virus(HPV) with cervical cancer has been supported by multiple lines of evidence. Therefore, in the case of dysplasia, the presence of HPV-DNA should be detected and its subtypes identified. This is important in the determination of the prognosis for cervical disease. We reported a study in which the localization and types of HPV in cervical diseases was identified by in situ polymerase chain reaction(PCR), using biotin-labelled DNA probes. The in situ PCR, used by us was modified of Nuovo's method. We used biopsy materials of 18 CIN and 9 SCC cases(total 27 cases), all of which had been detected HPV-DNA by Southern blot hybridization, but not detected by in situ hybridization. A positive intranuclear reaction was detected in 13 of 18 CIN cases and 6 of 9 SCC cases(total 19 positive cases). Molecular biological techniques are the most reliable methods for detecting specific tumor genes and virus DNA. In situ hybridization has the advantage of enabling recognition of the cellular localization of the DNA in histologic specimens, but its sensitivity in inferior to the other techniques such as Southern blot, Dot blot and PCR. In situ PCR method possesses the advantages of both PCR and in situ hybridization in being highly sensitive and enabling visualization of the cellular localization of the DNA. In our present study, we succeeded to detect HPV-DNA in cervical biopsies of CIN and SCC cases by the in situ PCR.

[Detection of papilloma virus of the human uterine cervix by in situ hybridization method--comparison with immunohistochemistry].

The usual methods for pathological diagnosis of HPV infection of the uterine cervix include screening in cytodiagnosis and histodiagnosis and confirmation by immunohistochemistry (IHC) method. However, some institutes have recently begun to use in situ hybridization (ISH) method for definitive diagnosis using a DNA probe. We compared IHC with ISH with regards to the localization and rate of detection of HPV in lesions of the uterine cervix such as dysplasia and squamous cell carcinoma in the present study. The cases found positive by IHC showed brownish nuclei of the epithelium and those positive in ISH showed purple to purplish-black nuclei. The comparison of cases positive by both methods revealed that the number of cells positive by IHC was smaller than that by ISH, and the cells positive by IHC were localized in the superficial layer. HPV was detected by the IHC various lesions of the uterine cervix in 13 (12.3%) of 106 patients, while it was detected by the ISH in 39 (36.8%) of 106 patients. The results of both methods were in accordance in 66.0% (77 patients; positively in 8 and negatively in 62). The detection sensitivity of IHC is lower than that of ISH. IHC cannot be used to identify the type of HPV, and it is impossible to confirm the presence or absence of virus by this method in cases of malignant changes. ISH is therefore necessary for identification of HPV and investigation of a histopathological relationship between HPV type and malignant change.

[Detection of papilloma virus in the cervix of the human uterus by in situ hybridization--Comparative study of detection rate by Southern blot method].

Cases found to be positive for human papilloma virus (HPV) infection in the cervix by Southern blot method were evaluated with a newly developed kit for in situ HPV tissue hybridization. By the in situ hybridization method, HPV-DNA was detected without damaging the tissue structure. It appeared as purple or black stains in the nucleus of the epithelial cells that were located at the level of one-third of the epithelial thickness from the surface. The cases that were negative with the Southern blot method were also negative with the in situ hybridization method, but only 55.8% of the cases positive with the Southern blot method were positive with the in situ hybridization. Although in situ hybridization method is not as sensitive as the Southern blot method, it allows analysis of old paraffin blocks and comparison with pathological features.

[Detection of HPV-DNA in the various uterocervical lesions by in situ hybridization].

We detected HPV (Human Papilloma Virus) -DNA in various uterocervical lesions by in situ hybridization using biotinylated DNA probes. In cases positive for HPV, the nuclei of the epithelial cells was purple to blackish purple. In 2 of 6 cases of chronic cervicitis, HPV-DNA was detected in the outer layer of the squamous epithelium. Eleven of 19 with mild dysplasia (57.9%) showed a positive reaction in the upper one-third of the epidermis in a mainly consisting of koilocytotic cells. All 6 patients with moderate dysplasia had positive cells among the koilocytotic cells and atypical cells in the middle layer. Five of 11 patients with severe dysplasia had scattered positive cells. Two of them had atypical condylomatoid lesions. Eight of 32 patients with squamous cell carcinoma were positive for HPV-DNA, but there was no consistent distribution pattern of the positive cells.

[Detection of HPV infection in CIN and invasive cancers by in situ hybridization method--comparison with histopathology].

The causal association of human papilloma virus (HPV) with cervical cancer has been supported by multiple lines of evidence. Therefore, in the case of dysplasia, the presence of HPV-DNA should be detected and its subtypes identified. This is important in the determination of the prognosis for cervical disease. We reported a study in which the localization and types of HPV in cervical diseases was identified by in situ hybridization using biotin-labelled DNA probes. Seven types (3 basic forms) of HPV were used as DNA probes. HPV types used were following: 6/11, 16/18, 31/33/35. In this study, we introduced 7 new types (3 forms) of probes for a total of 14 types (6 forms). The new probes introduced were 42/43/45, 45/56, 51/52. Using these probes, the rate of detection HPV according to types was examined. In addition, localization of HPV infection and its relationship with histopathological findings of cervical disease were evaluated. 1) Types 6/11 and 45/56 were found in the lesions with less important histological findings (CIN I). Types 16/18, 31/33/35, 51/52 were found in the all lesions. Type 42/43/44 were not detected in this study. 2) The addition of 7 new types (3 new forms) of probes resulted in a 17% increase (16 cases) in the rate of detection of HPV compared to our previous study. 3) Localization and distribution pattern of the HPV is not, as has been reported so far, dependent on HPV type but rather on the histological characteristics, such as the degree of dysplasia.

[Cytopathology and histopathology of human papilloma virus].

Human papilloma viruses (HPV) have been regarded seriously not only as an important agent causing sexually transmitted disease, but also because of its association with malignant transformation. Over 70 types of HPV are known, of which 30 types have been detected in specimens from the cervix. These have been classified into a low risk group, a high risk group and an intermediate group according to their association with malignant transformation. Therefore, diagnosis of cervical HPV infection and to a certain extent the HPV type are extremely important. Diagnostic methods of HPV infection include morphological methods such as cytological, pathological and electron microscopical diagnoses; DNA hybridization methods such as Southern blot hybridization (SBH) and Dot blot hybridization (DBH); and a combination of the two methods such as in situ hybridization (ISH), in situ polymerase chain reaction (in situ PCR) and polymerase chain reaction-in situ hybridization (PCR-ISH). Morphological methods using koilocytosis as an indicator have a low detection rate for HPV. Although ISH, in situ PCR and PCR-ISH have lower sensitivity that SBH and DBH, they enable visual localization of the detected DNA in cells or tissues. These methods are therefore important for cytopathologists and histopathologists. In this study, we report the morphological characteristics of HPV, as well as the advantages, disadvantages and detection rates of the various diagnostic methods.

[HPV infection of the uterine cervix by cytodiagnosis, histodiagnosis and dot blot hybridization method].

Human papilloma virus (HPV) infection is diagnosed by morphological methods (including cytodiagnosis, histodiagnosis and electron microscopy) and DNA diagnostic methods. One of the DNA diagnostic methods, Dot blot hybridization (DBH), is generally regarded to be superior to the morphological methods in terms of specificity and sensitivity. There have recently been reports showing that such morphological methods as cytodiagnosis [for koilocytotic atypia (KC) or koilocytosis] and histodiagnosis are best for HPV detection in laboratories because they have better sensitivity, and are cheaper, and procedurally simpler than the DBH method. We compared detection rates by morphological methods (cytodiagnosis and histodiagnosis) and the DBH method. (Materials and Methods) The subjects were 377 patients who came to the Department of Obstetrics and Gynecology of Juntendo University Urayasu Hospital and had abnormal findings in the uterine cervix by cytodiagnosis of colposcopy. Uterine cervix swabs were scraped during colposcopic examination, and specimens for Papanicolaou and Giemsa staining were prepared on 2 slides. Scraping was again performed for the DBH (Vira pap. and type, Toray Industries, Inc.) method using swabs specified for the kit, and cells were obtained in a collection kit. At the same time, at least 3 sites including, possible atypical conversion zones, were biopsied, followed by paraffin embedding and hematoxylin-eosin staining of the specimens. (Results) 1) Comparison of detection rates by cytodiagnosis and the DBH method The positive rates by the DBH method were determined according to cytodiagnostic category in 377 patients. HPV-DNA was detected in every category and in 86 (22.8%) of all of the patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of taurine on the ATPase activity in the human erythrocyte membrane.

Effects of taurine on the total ATPase activity inhibited by ouabain were investigated in the human erythrocyte membrane. The enzyme activity was not activated by taurine in the absence of calcium, but was significantly activated by taurine (15-60 mM) in the presence of calcium (5 mM) by which the enzyme is inhibited. Since taurine does not form a chelate complex with calcium ion, this activation may be due to an action of taurine in the presence of calcium through competition with these ions on the membrane.

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture.

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N'-bis[N″, N‴-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.

Cardiac rhabdomyoma of a neonate: an autopsy case report.

A primigravida delivered a cyanosed female infant with a very low Apgar score. Cardiac anomaly of the fetus was detected at 32 weeks of gestation by ultrasonography. The baby died on the day of delivery. Autopsy revealed multiple tumor masses in the interventricular septum and ventricular walls. The tumor originating from the interventricular septum was the largest and measured 3.7 x 3 cm. Histologically, the tumor was composed of large polygonal glycogen-laden cells and 'spider-cells'. Eosinophilic giant histiocytic cells were also observed in the spleen. Ultrastructural features of the tumor cells correlated with those of typical cardiac muscle cells.