In immunofluorescence microscopy (IFM), the repression of out of focus fluorescence signal is crucial in order to obtain high-resolution images. One option to acquire high vertical resolution (z-axis resolution) is to produce optical sections with a confocal microscope. The z-axis resolution usually obtained with confocal microscopy of biological samples is about 500 nm. Another option is to produce very thin sections with a cryo-ultramicrotome (physical sections). The ultrathin cryosections we employ are about 100 nm in thickness: thus all of the fluorescence must come from within this 100 nm thickness. The use of ultrathin cryosections permits the acquisition of extremely high-quality images and minimizes the possibility for false localization in IFM (Fig. 1). Ultrathin cryosections can be applied to immunoelectron microscopy (IEM) as well as IFM (Fig. 2). We show new methods of ultrathin cryosection immunocytochemistry^1-3. Human full-term placentas were fixed with 4% paraformaldehyde, solidified with 10% gelatin, infiltrated with 2.3 M sucrose, and then frozen in liquid nitrogen. Ultrathin cryosections were cut with a cryo-ultramicrotome and then transferred to glass cover slips for IFM or to nickel grids for IEM. Cryosections were incubated with mouse anti-p230, a trans-Golgi network marker, and subsequently incubated with Alexa 488-labeled goat anti-mouse IgG or with goat anti-mouse 5-nm colloidal gold particles. For visualization and preservation of ultrastructure of cryosections at the electron microscopic level, the sections on grids were postfixed with ferrocyanide-reduced osmium and then stained with uranyl acetate and lead citrate in polyvinyl alcohol¹. Ultrathin cryosection immunocytochemistry should be an important technique for functional genomics research, especially for the analysis of the in situ expression of target molecules^2,3.

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Miscarriage is the most common complication of human pregnancy. Although the causes of recurrent miscarriage (RM) are various, immunologic aberrations between mother and fetus might be one of the causes of miscarriage. Immune responses fall into two categories: autoimmune and alloimmune. Currently, no appropriate diagnostic method has been established to identify alloimmune causes. We observed that NK cell activity in women with RM was higher than that in women without a history of miscarriage. Functional or quantitative analysis of NK cells could be used to identify alloimmune causes. Paternal lymphocyte immunization has been the most widely used treatment for alloimmune-mediated miscarriages. However, the latest Cochrane review by Scott reached the current conclusion that lymphocyte immunization therapy provided no significant beneficial effect over placebo in preventing further miscarriage. Approximately 70% of Japanese university hospitals are still performing paternal lymphocyte immunization. Is paternal lymphocyte immunization a relic of the past? Randomized controlled trials based on adequate patient selection in Japan should provide an answer to this question.

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Background: The Functional Assessment of Cancer Therapy-Anemia (FACT-An) questionnaire, which consists of a core questionnaire named the General Measure of the Functional Assessment of Cancer Therapy (FACT-G) and the Anemia additional concerns subscale, was developed in an English-speaking culture. The validation of the Japanese FACT-G was reported previously (Fumimoto et al., 2001), and, in this report, a cross-cultural validation for the subscale was performed. Methods: The Japanese version was developed through an iterative forward-backward translation sequence used throughout the Functional Assessment of Chronic Illness Therapy (FACIT) Multilingual Translation Project. In evaluating psychometric performance, its construct validity was investigated by exploratory factor-analyses, and confirmed by Cronbach's alpha coefficient. Results: The FACT-An was given to 180 patients with lung cancer. Using the 20 items of the Anemia subscale, a factor analysis extracted four factors of fatigue, chest condition, activities and headache. When analyzed as two extracted factors, fatigue, chest condition and headache were combined to be a major factor, although the minor factor of activities still remained. Thirteen of the 20 items construct the Fatigue additional concerns subscale. Cronbach's alpha coefficients for the Fatigue subscale (0.93) and the Anemia subscale (0.88) confirmed that, although these subscales had items that focus on different aspects of anemia or fatigue, each subscale was unidimensional. Clinical validity was indicated by moderate values of Spearman's correlation coefficients between Eastern Cooperative Oncology Group performance status rating (ECOG PSR) and the Anemia subscale (-0.50) or the Fatigue subscale (-0.48). Conclusion: Both the Fatigue subscale and the Anemia subscale are valid in Japan, indicating that FACT-An is an instrument that is applicable across cultures and particularly with a Japanese cancer population.

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Over a five-year period, we reviewed 19 fetuses who were prenatally diagnosed with congenital heart disease, including hemodynamically significant arrhythmias. Five of them had fetal tachyarrhythmias, and 14 had structural heart disease. The outcomes were: six intrauterine deaths, five neonatal deaths, and three infant surgeries. Six of the fetuses had chromosomal abnormalities, four had extracardiac anomalies, and two had hydrops fetalis. Of the 96 neonates with congenital heart disease found during the study period, the overall detection rate was 20%; 16% of the neonates with structural cardiac defects and 83% of the neonates with arrhythmias. Some of the complex cardiac defects with normal fetal four-chamber view were difficult to detect prenatally. During the course of the pregnancy, 37% of the fetuses with prenatally diagnosed congenital heart disease were found to have intrauterine growth retardation, and 26% were found to have an abnormal amniotic fluid volume. In view of our findings, a comprehensive screening system should be more frequently considered in order to improve both detection rate and perinatal management.

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Objective: Sensory disturbance due to excision of the great auricular nerve in patients who have undergone parotidectomy sometimes causes discomfort to the patients. In order to reduce the postoperative discomfort of the pinna, we tried to preserve the posterior branch of the great auricular nerve. Methods: Forty patients with parotid tumor were included in this study. Twenty-one of these patients had pleomorphic adenoma, 16 had adenolymphoma and 3 had a low grade malignant tumor. Sensations of the pinna and the quality of life (QOL) after parotidectomy were evaluated using a 0-100 Visual Analogue Scale (VAS) assessed at 2 weeks, 1 month, 2 months, 3 months and 6 months after parotidectomy. Results: The posterior branch of the great auricular nerve was preserved in 26 out of 40 patients (65%). No difference was observed in the incidence of complications except sensory disturbance of the pinna with this surgical procedure as compared to the surgical technique where the great auricular nerve was excised. The VAS score for the sensation was significantly higher in the group of patients whose great auricular nerve was preserved at 2 months (35.0±20.8 vs. 18.5±9.2), 3 months (64.4±18.3 vs. 26.4±13.8) and 6 months (66.9±16.2 vs. 26.6±11.4) after parotidectomy. The VAS score for the QOL was also significantly higher in the group of patients whose great auricular nerve was preserved at 2 months (50.3±21.8 vs. 35.1±14.5), 3 months (69.5±27.5 vs. 45.9±22.6) as well as 6 months (71.9±24.1 vs. 45.7±19.1) after parotidectomy. Conclusion: Preservation of the posterior branch of the great auricular nerve during parotidectomy is valuable in order to reduce the postoperative sensory disturbance of the pinna that follows conventional surgery. It further helps to improve the QOL of these patients after parotidectomy.

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We report a case of pulmonary aspergilloma in a 27-year-old woman with anorexia nervosa who underwent a video-assisted thoracic surgery (VATS) lobectomy. On admission, she had lost 38% of her original weight but the laboratory data were normal. She had refused treatment for anorexia nervosa for nine years ago and may have been predisposed to opportunistic conditions. The aspergilloma was developed in a simple bulla formed in the course of healing of a lung abscess and a VATS lobectomy was safely and cosmetically performed. Wedge resection was difficult due to the size of the lesion. The residual lobes expanded very well and the postoperative course was uneventful. VATS is considered to be an efficient method in the treatment of pulmonary aspergilloma in patients with better lung function and localized pulmonary disease.

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Spontaneous perforation of a duodenal diverticulum is a rare but serious complication with significant mortality rates. Just over 100 cases have been reported in the world literature. One case of perforated duodenal diverticulum treated by simple closure is reported. An elderly female was admitted to our hospital with an acute abdomen. Computed tomography of the abdomen showed retroperitoneal air around the duodenum and right kidney. Laparotomy with a Kocher maneuver disclosed a perforated diverticulm in the second portion of the duodenum. Although diverticulectomy is the most common treatment, simple closure of the perforated duodenal diverticulum with drainage was performed to avoid injury to the distal common bile duct and ampulla of Vater, which were close to the diverticulum. The patient has fully recovered and has been asymptomatic with no signs of recurrence for 25 months.

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Postoperative endophthalmitis is one of the most serious complications after cataract surgery though its frequency may be low. We report a case with post-cataract extraction bacterial endophthalmitis treated favorably by core vitrectomy through pars plana with anterior vitrectomy cutter (A-vit). The patient, a 72-year-old woman, presented with blurred vision 7 days after phacoemulsification and aspiration (PEA) and intraocular lens (IOL) implantation. Her initial visual acuity was counting fingers. As hypopyon and corneal edema progressed in a few hours, we decided to perform vitectomy. Firstly, we performed IOL explantation and anterior vitrectomy through the corneal stab incision with A-vit attached to the phaco machine. The inflammation, however, appeared to be severe. Secondly we performed core vitrectomy with the same cutter as we used in the first operation through pars plana as well as intravitreal injection of vancomycin on the following day. The inflammation was gradually subsided and her corrected visual acuity was recovered to 30/20 at 7 months after the vitrectomy. The results is suggest that for cataract surgeons in the facilities that are not equipped with 3-port vitrectomy machine, post-cataract extraction bacterial endophthalmitis of the emergency stage can be successfully treated by core vitrectomy through pars plana as well as intravitreal injection of antibiotics with neither vitreous shaving at the vitreous base nor artificial posterior vitreous detachment.

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Objectives: To analyze our hospital laboratory microbiological data by using WHONET 5-Microbiology laboratory database software-, and to acquire information about antimicrobial resistance of Staphylococcus aureus strains among every ward. Materials and methods: The database of Staphylococcus aureus strains had been brought to our hospital microbiology laboratory from every ward in our hospital from September 2001 till December 2002. Analysis was performed under the condition as one isolate per one patient. Starting of 'resistance profile' analysis in WHONET 5 and analyzing the microbiological laboratory testing reports for every ward. We chose Oxacillin, Levofloxacin, Erythromycin and Gentamicin as the antimicrobials that need to be investigated for resistance. We evaluated the monthly transition of resistance ratios with regard to the specific wards that have the moving lines of inpatients in order to verify the hypothesis that resistant strains may be carried from ward to ward along the moving lines of inpatients. Results: The data of 2,113 Staphylococcus aureus strains were accumulated and analyzed. Overall Oxacillin resistance ratio in our hospital was 65.7%. The ward of the smallest Oxacillin resistance ratio was Pediatrics/Ophthalmology ward. The ratios of Oxacillin resistant were varied as from 67.9% to 96.7% regardless the categories of wards such as internal medicine or surgery. Multi-resistant MRSA strains were overwhelmingly dominant in the wards of surgery. The ratios of the Gentamicin sensitive strains that were resistant to Oxacillin were high over the every ward. The moving lines of inpatients existed between ICU/CCU ward and three rear wards. Two rear wards whose Oxacillin resistance changes were reflected to those of ICU/CCU, but one rear ward was not. Conclusion: Variation of resistant degree among wards were very obvious and large. We could survey the wards where patient-to-patient transmission of resistant organisms might occur along the moving lines of inpatients. WHONET 5 will be recognized as an analysis and surveillance tool for every infection control team to survey the suspicious wards.

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The purpose of this study was to determine whether use of an air ambulance service using a helicopter with a critical care physician and nurse on board (doctor helicopter service; DHS) could shorten the prehospital delay. We evaluated the initial treatment time and the transport time in 30 patients transported by DHS and 30 patients transported by ground ambulance service (GAS). The initial treatment time was significantly shorter in the DHS group (11.3+/-5.4 min) than in the GAS group (29.5+/-15.3 min). But the transport time in the DHS group (26.1+/-8.6 min) was not different from that in the GAS group. The difference in the initial treatment time was remarkable for patients transported from distant areas (12.7+/-5.6 min for DHS, and 42.1+/-13.8 min for GAS) and DHS shortened the initial treatment time by 30 min compared with GAS. The transport time was shorter for DHS (30.5+/-9.9 min) than for GAS (42.1+/-13.8 min) for patients transported from distant areas, but it was not significantly different for patients transported from nearby areas (22.3+/-5.0 min for DHS, and 18.4+/-2.4 min for GAS). In conclusion, DHS is important in the management of life-threatening cardiovascular diseases, and has a significant impact when GAS cannot transport a patient to the hospital within 20 min.

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