Microscopic Verification Research Articles

Geographic mapping of head and neck cancers in Nanded, Maharashtra, India: A hospital based study

Background: Head and neck cancers are most common in developing countries. It is the most common cancer of males in India and fifth in females. India is classified as a lower middle income group country by World Bank. Nanded, Maharashtra, India is a district place where this government medical college and hospital is situated; this is a tertiary medical care centre and caters nearby districts like Parbhani, Hingoli, Yavatmal and Adilabad. Methods: This is cross sectional study of 18 months. Cases are selected with superficially palpable head and neck lesions referred to Cytology OPD, and hospital ward with clinical diagnosis of any head and neck neoplastic lesions. An FNAC smear in the Cytology section of CCL and in ward finally the microscopic verification is done by biopsies with histopathological confirmation is done and confirmed cases are included in this study. Geographical mapping done by making the master chart of patient’s permanent addresses. Patients categorized as per codes by NCRP used for Atlas of cancer of India. Results: Relative proportion percentage of head and neck cancers is 88.33 were from Nanded district, followed by Hingoli, Parbhani, Yavatmal from Maharashtra state and Adilabad districts from Telangana state. Conclusions: This centre caters more than five districts including Maharashtra and Telangana state, in this hospital based ‘head and neck cancer registry’ of microscopically confirmed cancers, maximum cases are from Nanded district. This institute does not cover the geographic area mentioned in the literature of highest and lowest relative proportion percentage of head and neck cancers.

Retrospective analysis of the superficial dermatomycosis prevalence in areas of the Greater Caucasus of Azerbaijan

Aim. Study the epidemiological situation regarding the prevalence of skin, hair or nails superficial mycoses in 15 districts of the Greater Caucasus of Azerbaijan for the period from 2000 to 2012.
 Methods. Such indicators as the number of patient visits, periodic screening examinations and admissions were analyzed using the current and archived medical records of the Municipal Center for Skin and Sexually transmitted diseases №1, Republican Center for Skin and Sexually transmitted diseases, Republican Paediatric Center for Skin and Sexually transmitted diseases №3 of the Azerbaijan Republic. Skin superficial mycoses were diagnosed after laboratory (microscopic) verification of fungal mycelium presence. Intensive indicators were calculated, such as the prevalence of skin superficial mycoses and the number of patient visits due to skin superficial mycoses.
 Results. The prevalence of the skin superficial mycoses has increased in the Greater Caucasus of Azerbaijan area at the examined period (2000 to 2012) since 2004, with the prevalence peaks in 2007, 2009 and 2011. Men were twice (61.54%) more commonly affected compared to women (38.06%). The highest prevalence of skin superficial mycoses was registered in age groups of 0-10 (38.69%) and 11-20 (20.83%) years, the main diagnosis were «scalp mycosis» (27.98%) and «tinea versicolor» (22.62%). The prevalence of skin candidiasis (1.19±0.84%), onychomycosis (4.17±1.54%), tinea cruris (5.36±1.74%), combined scalp and glabrous skin mycosis (5.95±1.83%), athlete’s foot (8.93±2.20%), «Kerion» lesions (10.71±2.39%), glabrous skin mycosis (13.10±2.60%) increased. The prevalence of skin superficial mycoses was the highest in 2011 - 1.980±0.388%, the number of patient visits due to skin superficial mycoses - 0.712±0.140%; in 2007 the following numbers were 1.911±0.390% and 0.607±0.124% respectively, in 2009 - 1.637±0.357% and 0.537±0.117%, duplicating the prevalence peaks. High prevalence of superficial dermatomycoses was seen in Khizi and Ismailli Districts, the lowest - in Balakan, Qusar, Oghuz, Shaki Districts. Conclusions. In the current social and economic conditions, the system of complex examination (cultures, microscopy) of patients with skin mycoses is required, as well as the program of targeted preventive measures and improvement of medical and social aid management.

대전광역시와 충청남도의 13년간(2000-2012) 대장암 발생 추세

Objectives: Colorectal cancer is one of the major cancers in South Korea. We described the time trends in colorectal cancer incidence in Daejeon, a metropolitan city, and Chungcheongnam-do (Chungnam), a rural province, South Korea. Methods: Using the databases from the Daejeon Cancer Registry (DCR) and the Chungnam Cancer Registry (CCR), age-standardized (to world standard population) rates for incidence (ASRW) were calculated. Average annual percent change (AAPC) was assessed as a trend indicator. The completeness (such as the mortality/incidence ratio) and validity (such as the death certificate only %, microscopic verification %, primary site uncertain %, and age unknown %) were analyzed to examine the data quality of DCR and CCR. Results: Incidence of colorectal cancer showed increasing trend in both sexes. Over the years 2000-2012 in Daejeon, ASRW was increased significantly from 37.2 to 51.7 per 100,000 person-years (AAPC, 3.9%) among men and from 17.1 to 28.4 (AAPC, 3.9%) among women, respectively. In Chungnam, ASRW was also increased from 29.8 to 50.1 per 100,000 person-years (AAPC, 5.1%) among men and from 15.9 to 26.6 (AAPC, 3.2%) among women, respectively. The AAPC for colon cancer was greater than rectal cancer in both Daejeon and Chungnam. The trend of rectal cancer incidence was differ by sex (AAPC in men vs women, 2.7% vs 1.7% in Daejeon; 3.5% vs 0.8% in Chungnam). Indices of completeness and validity showed that the quality control of DCR and CCR was adequate to describe the trends of ASRW. Conclusions: Both Daejeon and Chungnam have had a rapid increase in colorectal cancer incidence. Monitoring and intervention are required on the risk factors which may contribute to this trend.

Incidence of cancer in children aged 0–14 years in Taiwan, 1996–2010

Studies have found lower risk of childhood cancer among Asian children. We aim to characterize the recent incidence and incidence-trend of childhood cancer in Taiwan after the National Health Insurance program was launched in March 1995. Data were extracted from the Taiwan Cancer Registry, a population-based database established in 1979. Cases diagnosed at age 0–14 from 1996 to 2010 were analyzed and categorized according to the International Classification of Childhood Cancer, Third Edition (ICCC-3). In total, 8032 childhood cancer cases were included, with a microscopic verification rate of 93.9%. The overall age-standardized rate (ASR) of incidence adjusted to the 2000 World Standard Population is 125.0 cases/million, with a male-to-female ratio of 1.3. The top five cancer types (ICCC-3 subgroup[s]; ASR per million) are acute lymphoblastic leukemia (Ia, 30.3), acute myeloid leukemia (Ib; 9.4), non-Hodgkin lymphoma (IIb,c,e, 9.0), extracranial germ cell tumor (Xb,c; 8.3), and neuroblastoma (IVa; 7.8). The median age of diagnosis was 6 years for both genders. During the study period, the ASR of childhood cancer has been increasing at a rate of 1.2% per year (95% confidence interval, 0.6–1.7%). In contrast to Western countries, China, Japan, and Taiwan have lower incidence of childhood cancer; however, Taiwan's incidence rates of childhood germ cell tumors and hepatic tumors are higher. In conclusion, this population-based study reveals that the incidence rate of childhood cancer in Taiwan is rising consistently. The high incidence of germ cell tumors warrants further investigation.

Braiding non-Abelian quasiholes in fractional quantum Hall states.

Quasiholes in certain fractional quantum Hall states are promising candidates for the experimental realization of non-Abelian anyons. They are assumed to be localized excitations, and to display non-Abelian statistics when sufficiently separated, but these properties have not been explicitly demonstrated except for the Moore-Read state. In this work, we apply the newly developed matrix product state technique to examine these exotic excitations. For the Moore-Read and the Z_{3} Read-Rezayi states, we estimate the quasihole radii, and determine the correlation lengths associated with the exponential convergence of the braiding statistics. We provide the first microscopic verification for the Fibonacci nature of the Z_{3} Read-Rezayi quasiholes. We also present evidence for the failure of plasma screening in the nonunitary Gaffnian wave function.

Quality of information and cancer care planning in China: a commentary to the report of cancer incidence and mortality in China.

Quality of information and cancer care planning in China: a commentary to the report of cancer incidence and mortality in China.

Microscopic verification of sensitive circuits of a quartz resonator applied in dew point recognition

This paper is aimed to carry out experimental verification of sensitive circuits of a quartz resonator applied in dew point recognition and it is proven at a microscopic level to be feasible in dew point recognition with great reliability and accuracy by using the high speed video. The sensor based on the sensitive circuits of a quartz resonator is composed of a quartz crystal and a Peltier module. Proactive approach is taken to produce condensation on the surface of the quartz crystal. The electrical parameters of the quartz crystal is changed due to dew condensation and then the resonant circuit stops oscillating. Initially, it needs to analyze the oscillation circuit and electrical characteristics of the quartz crystal, and then take photos of the surface of quartz electrodes with the high speed camera and microscope. Gray statistics is made for images of dew formation in its whole process. When the statistical results are analyzed in comparison to oscillation of the circuit, it shows that the time of the circuit oscillation stopping is roughly consistent with the time when moisture condensation occurs on the surface of the quartz electrode. And the time error of the two phenomena is less than 0.5 s, which is much less than response time of the platinum resistance sensor to the temperature. Furthermore, the results provide a strong theoretical basis for further measurement of the dew point temperature by this recognition method.

전라북도의 10년간(2001~2010) 암 발생률 추이 및 암등록 자료의 질 관리 지표 분석

본 연구에서는 전라북도 지역암등록본부로부터 2001년부터 2010년까지 10년간 수집된 암등록 자료를 토대로 전라북도 지역의 성별, 연령군별, 호발암별 암발생률 비교 분석과 변동 추이를 살펴보았으며, 또한 국제암연구소에서 제시하는 평가 지표를 기반으로 암등록자료의 질적 수준을 평가하였다. 전라북도 지역의 암발생률 통계 및 암등록 자료의 질 관리 지표는 전북지역암센터의 지역암등록본부로부터 수집되었다. 모든 암들에 대한 성별조발생율(CR)과 연령표준화발생률(ASR)의 10년간 추이를 살펴보았으며, 남녀 5대 호발암에 대한 ASR의 10년간 추이를 분석하였다. 또한 전라북도 지역암등록본부의 암등록 자료에 대한 년도별, 성별, 호발암별 질적 수준을 평가하기 위해 충실도와 타당도를 분석하였다. 충실도 평가를 위해 사용된 지표는 사망/발생비(MI%)와 연령별 발생률 곡선이며, 타당도 평가를 위해 사용된 지표는 연령 미상률(UNK%), 현미경적 확진율(MV%) 그리고 사망진단서에서만 확인가능한 환자의 분율(DCO%)이다. 암 발생률의 10년간 변동 추이는 joinpoint 회귀분석 도구를 이용하였으며, 해당 기간에 대한 연평균 암발생 변화율(AAPC)을 측정하였다. 10년간 전라북도 지역에서의 남성 호발암 순위는 위암이 22.2%로 가장 높았으며, 다음으로 폐암 16.6%, 대장암 12.8%, 간암 12.3%, 전립샘암 6.2% 순으로 나타났다. 여성에서는 갑상샘암이 17.8%로 가장 높았고, 위암 14.7%, 유방암 11.6%, 대장암 11.5%, 폐암 7.7% 순으로 나타났다. 10년 동안 모든 암들에 대한 연령표준화발생률(ASR)을 살펴보면 전체적으로 36.5% 증가하였다. 성별에 따른 ASR 증가율을 살펴보면 남성에서는 13.7%, 여성에서는 68% 증가하였다. 성별 호발암의 연간 변동 추이를 살펴보면 남성의 위암, 폐암, 간암 발생률은 각각 연평균 1.8%, 0.8%, 3.2% 감소하는 추세를 보이고 있으나, 대장암과 전립샘암은 각각 4.9%, 15.1% 증가하는 추세를 보였다. 여성에서는 5대 호발암중 유일하게 위암이 연평균 0.6% 감소하는 추세를 보였으나, 유방암, 대장암, 폐암은 각각 연평균 5.6%, 2.7%, 2.2% 증가 추세를 보였고, 특히 갑상샘암은 연평균 29.6%의 급격한 증가 추세를 보였다. 암등록 자료의 질적 수준 평가에서 충실도 평가 지표인 MI%는 남성에서 2001년 39.4%에서 2010년 32.4%로 낮아졌고, 여성의 경우 2001년 32.2%에서 2010년 23.5%로 낮아져 적정한 수준을 유지하였다. 타당도 평가 지표인 UNK%는 전라북도 지역에서 10년간 남녀 모두 0%로 나타났으며, DCO%는 남성에서 2001년 5.6%에서 2010년 1.3%로 낮아졌고, 여성에서는 2001년 6.1%에서 2010년 1.8%로 낮아져 암등록 자료의 정확성이 향상되었다. MV%는 남성의 경우 2001년 76.3%에서 2010년 88.5%로 증가하였고, 여성의 경우 2001년 78.4%에서 2010년 90.8%로 증가하여, 남녀 모두에서 암진단의 정확성이 높아진 것으로 확인되었다. 전라북도의 10년간 암발생률 추이를 분석한 결과 남성보다 여성에서 암발생률이 증가하고 있으며, 특히 남성에서는 대장암과 전립샘암이, 여성에서는 갑상샘암과 유방암이 증가하고 있음을 확인하였다. 또한 전라북도 지역암등록본부의 암등록 자료의 질관리 수준이 향상되고 있음을 확인하였다. 향후 연구에서는 대도시지역 또는 전라북도와 유사한 다른 지역과의 지역별, 연령별 호발암 및 암발생률 추이를 심층적이고 다각도로 비교 분석하여 향후 국가 차원의 암관리 대책 마련과 암 조기검진사업 추진 등의 보건 정책을 수립하는데 이와 같은 통계 자료가 반영될 수 있도록 해야 할 것이다. Objectives: This study was aimed to analyze the trends of cancer incidence and evaluate the quality control of cancer registration data in Jeollabuk-do from 2001 to 2010. Methods: Incidence data of all cancers and indices of quality of cancer registration data in Jeollabuk-do for the 10-year period were obtained from the Population-based Regional Cancer Registry in the Jeonbuk Regional Cancer Center. Trends in crude incidence rate (CR) and age-standardized incidence rate (ASR) for all cancers and incidence rates for major cancer sites by gender were analyzed. Joinpoint regression tool was used to describe and quantify trends. And the completeness and validity of cancer registration data were analyzed. Results: The major cancer sites in males were the stomach (22.2%), lung (16.6%), colorectum (12.8%), liver (12.3%) and prostate (6.2%), and in females were the thyroid (17.8%), stomach (14.7%), breast (11.6%), colorectum (11.5%) and lung (7.7%). Between 2001 to 2010, ASR for all cancers increased 13.7% in men, 68% in women, and 36.5% overall. ASR for all cancers increased by 1.2% per year in males and by 6.7% per year in females from 2001 to 2010. In the quality control of the cancer registration data between 2001 and 2010, death certificate only (DCO%) for men was decreased from 5.6% to 1.3% and DCO% for women decreased from 6.1% to 1.8%. Microscopic verification (MV%) increased in both men and women. And mortality/incidence ratio (MI%) declined in both men and women. Conclusions: The cancer incidence during the 10 years (2001-2010) in Jeollabuk-do was increasing especially for the colorectum and prostate in men, and for the thyroid and breast in women. The overall quality control of the cancer registry was gradually improving.

Single cell analysis exposes intratumor heterogeneity and suggests that FLT3-ITD is a late event in leukemogenesis

FMS-like tyrosine kinase 3 receptor-internal tandem duplication (FLT3-ITD) commonly occurs in acute myeloid leukemia and is considered rare in acute lymphocytic leukemia. Acute leukemia has poor prognosis, mainly due to relapse. Standard FLT3-ITD diagnostic techniques are based on genomic polymerase chain reaction and have recently incorporated GeneScan (Applied Biosystems, Foster City, CA) to identify variations of the FLT3 gene. As this is an average-based assay utilized in a heterogeneous leukemic cell population, we hypothesized that cells of acute leukemia, considered FLT3-ITD-negative by standard methods, could possess a fraction of FLT3-ITD-positive cells. The present study employed single cell mutation analysis to evaluate the FLT3-ITD status in newly diagnosed acute myeloid leukemia (n = 5) and acute lymphocytic leukemia (n = 3) patients. A total of 541 single leukemic cells and 36 mononuclear cells from healthy volunteers were analyzed. Seven patients, considered FLT3-ITD-negative according to bulk DNA analysis, appeared to possess a small fraction of FLT3-ITD-positive cells based on single cell analysis. Moreover, this approach revealed the heterogeneity of the tumor as evident by different FLT3-ITD mutations present in the same patient. The presence of a minor clone carrying FLT3-ITD in almost all patients tested provides evidence that this lesion is a common late event in leukemogenesis. Additionally, 3 relapsed patients demonstrated loss of heterozygosity of the normal allele, affecting 25%-100% of the cells found to be FLT3-ITD-positive. Though further clinical testing is warranted, these findings may have implications on the prognostic significance of FLT3-ITD and the use of targeted therapy.

The differentiation of the character of solid lesions in the breast in the compression sonoelastography. Part I and II.

I have carefully read the comments on two articles published by myself in issues 51 and 52 of “Journal of Ultrasonography,” concerning the differentiation of the character of solid lesions in the breasts in compression sonoelastography. I would like to thank Dr. Dominique Amy for his perceptive comments to which I shall try to relate. The application of a proper technique of ultrasound (US) examination and the usage of adequate terminology with respect to the assessed structures is undoubtedly significant. In my own research, I used standard radial technique of breast assessment and the visualized focal lesions were evaluated in two planes. Bearing in mind the terminology used, I agree that, histologically, there is no such a term as glandular tissue and that it indeed is a misnomer. It, however, became so strongly established in the standards of our US examination descriptions and reports that the usage of correct terms: epithelial and connective tissue might occur to be incomprehensible. This probably requires changing. With reference to the further part of the comment concerning the character of lesions categorized to group II (benign lesions), I must say that cystic lesions (anechoic, BIRADS-US 2, cysts) were not included in the examination. Only solid lesions, which manifested the features characteristic of BIRADS-US 3, 4 and 5, were taken into account. However, the microscopic verification revealed that approximately 57% of lesions in group II were fibrocystic. In the majority of cases, these lesions presented themselves as hypoechoic, one was hyperechoic and 14 were isoechoic and were accompanied by additional features characteristic of BIRADS-US 3 and 4. As far as the third remark is concerned connected with sonoelastography and sonoelastograms, I made every effort to perform the examinations in accordance with the guidelines proposed by the authors of the method (A. Itoh, E. Ueno), bearing in mind the limitations mentioned by Dr. Amy. Sonoelastographic techniques, due to their innovative character, are being continuously perfected. However, in the assessment of focal lesions, particularly those categorized as BIRADS-US 3, compression sonoelastography has already proven to be relevant which is confirmed by the guidelines published in “Ultraschall in der Medizin”/”European Journal of Ultrasound”(1). In the sonoelastograms published in the paper, I presented examples of focal lesions in the breasts with their surrounding structures on the basis of the guidelines published by Itoh and Ueno in “Radiology” in 2006(2). The compression level on all five presented sonoelastograms was identical (level 3). The FLR ratio, however, was presented in only one example elastogram for the purposes of illustrating the measurement technique. In both subject groups, FLR ratio was calculated for 94 out of 99 lesions. The size of lesions in both examined groups was analyzed in greater detail in the author's doctoral thesis (defended on 25 October 2012 in Cancer Centre, The Institute of Maria Sklodowska-Curie in Warsaw) – three measurements were analyzed in two perpendicular planes. The lesions in group I in all dimensions were greater than those in group II in a statistically significant way, which indeed, was only briefly mentioned in the commented publication. Unfortunately, malignant lesions are detected too late in Poland. Similarly, the rate of preinvasive carcinomas found in patients treated in Poland (4.4% in 2009 according to the Polish National Cancer Registry) is lower as compared to other countries of Western Europe or the USA. With reference to the comments concerning focality and centricity of breast neoplasms, I wish to explain that in group I, several patients manifested multifocal lesions (39 malignancies detected in 30 women). However, this issue was not analyzed in detail. What is more, in the part of the paper devoted to the discussion about false negative cases in elastograms, it was shown that in the subject group of malignant lesions (group I), there were cases of preinvasive and invasive ductal carcinomas (J Ultrason 2013; 13: 43). There were no cases of colloid (mucinous) carcinomas in the analyzed material. Thus, this type of a neoplasm was not discussed. I recognize the authority of Dr. Amy in the field of ultrasound assessment of the breasts, including the emphasized importance of having basic knowledge of anatomy as well as of pathophysiology and pathogenesis of breast carcinoma, and I would like to thank for a thorough analysis of my publication. I am particularly grateful for directing my attention to the need for using appropriate terminology both in everyday practice and in academic papers.

Uniformly thinned optical fibers produced via HF etching with spectral and microscopic verification

A method for producing uniformly thinned (etched) optical fibers is described, which can also be employed to etch optical fibers containing a Bragg grating (FBG) uniformly for evanescent-field-based sensing and other applications. Through a simple modification of this method, the fabrication of phase-shifted FBGs based on uneven etching is also shown. The critical role of how a fiber is secured is shown, and the success of the method is illustrated, by differential interference contrast microscopy images of uniformly etched FBGs. An etched FBG sensor for the monitoring of the refractive index of different glycerin solutions is demonstrated.

Identification of a Replication-independent Replacement Histone H3 in the Basidiomycete Ustilago maydis

Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ~25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.

Immunohistochemical Verification of Stereotactic Proton Radiosurgery Targeting Accuracy in the Rat Brain

We have developed a system for targeting functional areas in the rat brain with narrow proton beams. Due to the extremely sharp lateral dose falloff of high-energy proton beams, a cross-fire technique with multiple shoot-through beams converging on the target will be used to selectively irradiate small regions in the rat brain. The spatial accuracy of dose delivery is crucial and needs to be verified. This ongoing study intends to verify the accuracy and precision of the targeting method by developing immunohistochemical staining protocols based on IgG leakage through the radiation-induced breakdown of the blood-brain barrier (BBB) and various DNA damage markers. Male Sprague-Dawley (8 weeks, 150-190g) rats were subjected to hemispheric proton irradiation with 250 MeV proton beams at doses of 0 Gy, 8 Gy, and 25 Gy. IgG extravasation was used to show BBB damage from 4 to 72 hours after irradiation. Rat brains perfused with 4% paraformaldehyde-perfused were cryostat-sectioned and slide-mounted. Sections then underwent immunohistochemical staining using the ABC method (Santa Cruz Biotechnology). Radiation-induced H2AX phosphorylation, a sensitive marker of DNA double strand breaks (DSBs), was detected by Donkey anti-mouse IgG Alexa Fluor 488 (green), while nuclei were counterstained with propidium iodide (PI, red). After 25 Gy proton irradiation, radiation-induced BBB damage resulted in IgG extravasation as indicated by positive immunostaining for anti-IgG. Detection of γH2AX was also possible after hemibrain proton irradiation with 8 Gy. IgG staining is sensitive to local vascular radiation damage, which has the advantage of being maintained during longer time intervals (days to weeks rather than hours) compared to short-term DNA damage markers. The DSB marker γH2AX can be used for rapid confirmation of the targeted area. IgG staining and γH2AX immunofluorescences are useful for microscopic verification of the targeting accuracy of small-animal radiosurgery procedures with sub-millimeter resolution.

Calibration of the stereological estimation of the number of myelinated axons in the rat sciatic nerve: A multicenter study

Several sources of variability can affect stereological estimates. Here we measured the impact of potential sources of variability on numerical stereological estimates of myelinated axons in the adult rat sciatic nerve. Besides biological variation, parameters tested included two variations of stereological methods (unbiased counting frame versus 2D-disector), two sampling schemes (few large versus frequent small sampling boxes), and workstations with varying degrees of sophistication. All estimates were validated against exhaustive counts of the same nerve cross sections to obtain calibrated true numbers of myelinated axons (gold standard). In addition, we quantified errors in particle identification by comparing light microscopic and electron microscopic images of selected consecutive sections. Biological variation was 15.6%. There was no significant difference between the two stereological approaches or workstations used, but sampling schemes with few large samples yielded larger differences (20.7 ± 3.7% SEM) of estimates from true values, while frequent small samples showed significantly smaller differences (12.7 ± 1.9% SEM). Particle identification was accurate in 94% of cases (range: 89–98%). The most common identification error was due to profiles of Schwann cell nuclei mimicking profiles of small myelinated nerve fibers. We recommend sampling frequent small rather than few large areas, and conclude that workstations with basic stereological equipment are sufficient to obtain accurate estimates. Electron microscopic verification showed that particle misidentification had a surprisingly variable and large impact of up to 11%, corresponding to 2/3 of the biological variation (15.6%). Thus, errors in particle identification require further attention, and we provide a simple nerve fiber recognition test to assist investigators with self-testing and training.

Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability

The aim of the present study was to develop a flow cytometric procedure for the quantification of the proportion of viable, apoptotic, and necrotic polymorphonuclear neutrophilic leukocytes (PMNL) isolated from both low- and high-somatic-cell-count quarter milk samples. Milk PMNL were differentiated from other cells by indirect fluorescent labeling using a primary anti-bovine granulocyte monoclonal antibody (CH138A) and an Alexa 647-labeled secondary antibody. Polymorphonuclear neutrophilic leukocytes were identified flow cytometrically based on their cytoplasmic granularity and CH138A-positivity. Additional labeling with annexin-V-fluorescein isothiocyanate and propidium iodide was used to determine milk PMNL viability. Thirty milk samples were run in parallel to assess the repeatability of the immunoassay and 6 repeated measurements per sample were performed to assess the instrument stability. Fluorescence microscopic verification of the CH138A staining pattern showed both a high sensitivity (90.9%) and specificity (92.3%). The combination of the side-scatter properties of granulated PMNL and CH138A-Alexa 647 positivity allows the distinction of labeled PMNL from other milk cells and particles that may bind nonspecifically, and from autofluorescent particles present in milk. Quantification of the proportion of PMNL and viable, apoptotic, and necrotic subpopulations in parallel samples gave repeatable results with concordance correlation coefficients varying between 0.93 and 0.99. The average coefficient of variation for repeated measurements in identical samples ranged between 4.2 and 9.7%. In conclusion, this is the first flow cytometric method suited for the simultaneous quantification of viable, apoptotic, and necrotic bovine milk PMNL in a straightforward manner.

Anthropometry and Pancreatic Cancer Risk: An Illustration of the Importance of Microscopic Verification

Using data collected of a large population-based cohort study, we studied the association between anthropometric factors and the risk of pancreatic cancer. Furthermore, we investigated whether these associations differ among microscopically confirmed pancreatic cancer (MCPC) cases and non-MCPC (NMCPC) cases. The Netherlands Cohort Study on Diet and Cancer started in 1986 (120,852 men and women) and uses the case-cohort methodology. After 13.3 years of follow-up, 446 pancreatic cancer cases (of which 65% was microscopically confirmed) and 4,774 subcohort members were available for analysis. The multivariable incidence rate ratio of MCPC of men was 1.10 per increment of 1 kg.m(-2) (95% confidence interval, 1.04-1.18). Women had a rate ratio of MCPC of 1.08 (95% confidence interval, 1.03-1.13). Obese men [body mass index (BMI) >or=30 kg.m(-2)] had a 2.6-fold increased risk of MCPC compared with men with BMI 23 to 25 kg.m(-2). For women, this increase in risk was 1.7-fold. Change in BMI between age 20 years and baseline was also associated with MCPC in both men and women. In men and women, none of these associations were observed for NMCPC, with the exception of the increased risk for pancreatic cancer in obese men. We observed statistically significant associations between both BMI, gain in BMI, and pancreatic cancer risk. These associations are observed only in MCPC and not in NMCPC. If MCPC and NMCPC had been considered as one group, the reported associations would not have been detected. These findings stress the need to evaluate heterogeneity among pancreatic cancer cases in etiologic studies.

Automated detection and enumeration for toxic algae by solid-phase cytometry and the introduction of a new probe for Prymnesium parvum (Haptophyta: Prymnesiophyceae)

Harmful algal blooms have a severe impact on aquaculture and fishery and can be caused by toxic haptophytes and dinoflagellates. Different toxic species, which are not easy to distinguish from their morphologically similar and non-toxic relatives, occur in both groups. Sequencing of the large subunit ribosomal RNA of different strains and taxonomic relatives allowed the design of a probe specific to the toxic Prymnesium parvum spp. For the rapid detection and enumeration of Prymnesium and Alexandrium cells in cultures and environmental samples, respectively, protocols for fluorescence in situ hybridization were adapted for automated detection by a solid-phase cytometer, the ChemScan. This cytometer enables the automated counting of fluorescently labelled cells on a membrane filter and subsequently a microscopic verification of these results by the user, because the motorized stage of the microscope is driven to each positive signal by the computer software to localize that cell on the filter. With this fast detection method, it was possible to detect, enumerate and verify microalgal cells on a filter, with a detection limit of one cell per membrane filter.

Influence of the myocardial bridging phenomenon on the myocardial structure and the coronary arteries wall structure changes

Our research was performed to evaluate the influences of the myocardial bridgings of coronary arteries on the myocardial and coronary arteries wall structure chages, that could be a reason for multiple heart malfunctions. We analyzed the autopsy material, collected during a five-years period, and especially the group of 575 cases with the major aim to diagnose mors naturalis. In all cases with the presence of myocardial bridge over the arterial coronary wall revealed at autopsy, samples were taken for microscopic verification and examination. We found myocardial bridges over the coronary arteries or their major branches in 27 of the cases (4.70%). We believe that myocardial bridges compromise coronary perfusion by cyclic compression of the overbridged vessels, and that it could be the initial factor in the pathogenesis of arteriosclerotic degeneration processes on the coronary artery wall. We found different grades of arteriosclerotic changes in 88.89% of the cases, as well as fibrosis of myocardium in 88.89% and lipomatosis in 66.67% of the cases with the present myocardial bridges. Our results suggested that myocardial bridging of coronary arteries and/or their branches was the pathological and even lethal phenomenon that de serves more intensive clinical evaluation.

Small Angle Neutron Scattering Observation of Chain Retraction after a Large Step Deformation

The process of retraction in entangled linear chains after a fast nonlinear stretch was detected from time-resolved but quenched small angle neutron scattering (SANS) experiments on long, well-entangled polyisoprene chains. The statically obtained SANS data cover the relevant time regime for retraction, and they provide a direct, microscopic verification of this nonlinear process as predicted by the tube model. Clear, quantitative agreement is found with recent theories of contour length fluctuations and convective constraint release, using parameters obtained mainly from linear rheology. The theory captures the full range of scattering vectors once the crossover to fluctuations on length scales below the tube diameter is accounted for.

Adapting the CHEMTAX method for assessing phytoplankton taxonomic composition in Southeastern U.S. estuaries

CHEMTAX is a matrix factorization program used to derive taxonomic structure of phytoplankton from photosynthetic pigment vitios. The program was originally developed from and applied to the analysis of oceanic phytoplankton assemblages. We found that application of the original CHEMTAX reference matrix to southeastern United States estuarine systems produced inaccurate results, as verified by microscopy. Modification of the matrix, based primarily on the pigment ratios of 33 estuarine isolates, improved the predictive capabilities of CHEMTAX for our samples. Limitations of the method included an overstimation of diatom biomass (due to the inability to differentiate diatoms from taxa with chloroplasts derived from diatom endosymbionts, notably some dinoflagellates) and a tendency to exclude some raphidophyte species. In complement with microscopic verification, the method was shown to improve assessment of phytoplankton taxonomic composition.