Solid phase microextraction, mass spectrometry and metabolomic approaches for detection of potential urinary cancer biomarkers: a powerful strategy for breast cancer diagnosis

A sensitive assay to identify volatile organic metabolites (VOMs) as biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. Therefore the aim of this study was to establish the urinary metabolomic profile of breast cancer patients and healthy individuals (control group) and to explore the VOMs as potential biomarkers in breast cancer diagnosis at early stage. Solid-phase microextraction (SPME) using CAR/PDMS sorbent combined with gas chromatography–mass spectrometry was applied to obtain metabolomic information patterns of 26 breast cancer patients and 21 healthy individuals (controls). A total of seventy-nine VOMs, belonging to distinct chemical classes, were detected and identified in control and breast cancer groups. Ketones and sulfur compounds were the chemical classes with highest contribution for both groups. Results showed that excretion values of 6 VOMs among the total of 79 detected were found to be statistically different (p < 0.05). A significant increase in the peak area of (−)-4-carene, 3-heptanone, 1,2,4-trimethylbenzene, 2-methoxythiophene and phenol, in VOMs of cancer patients relatively to controls was observed. Statiscally significant lower abundances of dimethyl disulfide were found in cancer patients. Bioanalytical data were submitted to multivariate statistics [principal component analysis (PCA)], in order to visualize clusters of cases and to detect the VOMs that are able to differentiate cancer patients from healthy individuals. Very good discrimination within breast cancer and control groups was achieved. Nevertheless, a deep study using a larger number of patients must be carried out to confirm the results.

Avaliação dos parâmetros hematológicos e imunofenotípicos de pacientes com doenças linfoproliferativas crônicas no Rio Grande do Norte

Chronic lymphoproliferative disorders (DLPC) are lymphoid system diseases characterized by the abnormal proliferation of mature lymphocytes that affect B cells, T lymphocytes and NK cells. The aim of the study was to demonstrate the relevance of immunophenotyping by flow cytometry in patients with prolonged lymphocytosis and / or cytomorphological changes compatible with lymphoproliferative diseases. In this study 460 patients (244 men and 216 women) with DLPC were evaluated. Were analyzed by flow cytometry with a panel of monoclonal antibodies consisting of CD3, CD4, CD5, CD8, CD10, CD19, CD22, CD23, CD25, CD38, CD45, CD16/CD56, and HLADR heavy and light chains of immunoglobulins. It also examines information regarding age, gender of patients and laboratory data as leucocytes, cytomorphological analysis, platelet count and hemoglobin determination. The results showed 398 cases of chronic lymphoproliferative disorders and 62 of DLPC B cell lymphoproliferative diseases T. B showed the following distribution : 253 cases of chronic lymphocytic leukemia (CLL), 42 cases of multiple myeloma ( MM ), 37 cases of lymphoma non - Hodgkin lymphoma in leukemic phase (NHL) , 17 cases of pro- B lymphocytic leukemia ( B -PLL), 15 cases of mantle cell lymphoma (MCL ), 12 cases of plasma cell leukemia ( PCL), 9 cases of lymphoma Burkitt (Linf B), 8 cases of leukemia villous cells ( LCV), 3 cases of splenic lymphoma with villous cells (LECV), a case of follicular lymphoma (LF) and a Waldenströn macroglobulinemia ( MW). The diseases source NK / T were 23 cases of peripheral T cell lymphoma (LCTP), 14 cases of T prolymphocytic leukemia (T -PLL), 10 cases of leukemia T of large granular lymphocytes (LGL -T) 9 cases of leukemia cells of adult T (LCTA), 5 cases of Sezary syndrome (SS) and a case of large granular NK leukemia (LGL -NK) lymphocytes. In conclusion, the combined use of the monoclonal antibody panel careful cytomorphological analysis was shown to be essential in immune diagnosis and classification of chronic lymphoproliferative disorders. This study was approved by the IRB - HUOL under number 356 / 09

Photoacoustics as a tool for the diagnosis of radicular stress: Measurements in eucalyptus seedlings

In reforesting companies (cellulose industry), eucalyptus is usually cultivated in small plastic containers (50 mL). As seedlings remain for about 120 days in these containers-until transplantation-their roots become space restricted, with consequent limitations in water and nutrient absorption. These restrictions may lead to plant stress, decreasing productivity. In this work, we used the photoacoustic technique to evaluate the photosynthetic activity of Eucalyptus grandis, E. urophylla and E. urograndis seedlings subjected to this limited space availability, seeking a correlation with morphological parameters and fluorescence measurements in these seedlings. Photoacoustic, fluorescence, and morphological analysis were conducted every 15 days, from 45 to 120 days after sowing. Fluorescence and photosynthetic rate were evaluated in vivo and in situ, the latter one using the open photoacoustic technique. Data show that root dry matter diminished markedly at 90 and 120 days after sowing; this behavior showed a high correlation with the gas exchange component of the photoacoustic signal, as well as with the fluorescence ratio Fv/Fm. These results indicate that the soil volume of the container becomes insufficient for the roots after 90 days, probably leading to a nutritional deficiency in plants, which explains the decrease observed in the photosynthetic rate of seedlings. (C) 2003 American Institute of Physics.

Emprego da citometria de fluxo na avaliação do perfil imunofenotípico de pacientes com leucemia linfocítica crônica

Chronic lymphocytic leukemia (B-CLL) is a clonal proliferation of mature B lymphocytes characterized by indolent clinical course. Biologically this clonallity is characterized by low expression of surface immunoglobulin (sIg) with restriction to a single immunoglobulin light chain associated with high expression of CD5 antigen and positivity to B cell antigens lymphocytes such as CD19, CD20 and CD23 and negativity to FMC7. The immunological profile and morphological analysis of lymphoid cells are the main means for the differential diagnosis of B-CLL from other chronic lymphoproliferative diseases. The aim of this study was to evaluate the expression pattern of a variety of membrane antigens in leukemic cells originating from patients with B-CLL. In this study, peripheral blood samples from 80 patients with B-CLL were analyzed by multiparametric flow cytometry in addition to routine hematologic exams, using a panel of monoclonal antibodies (MoAb): CD45/CD14, CD3/CD19/CD45, CD4/CD8 / CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/CD19, CD1a, CD11b and also IgM/gD, kappa and lambda immunoglobulin light chains for the detection of surface immunoglobulin and clonal restriction for immunoglobulin light chain. The Hematological data were obtained from the hematological analyzer and cytomorphological analysis in blood film stained by Leishmann. The study samples consisted of 45 men and 35 women, ages ranging from 55 to 84 years (mean 65 years). Complete white blood count showed count ranging from 10.0 to 42.0 x 109/l. (mean 50.0 x 109/l) and lymphocytes count greater than 5.0 x 109/l in all cases. The neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/CD20+/HLADR+/CD23+) in the vast majority of the cases, associated to failed to stain for T cell markers (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 and FMC7. Leukemic cells of most patients also expressed low intensity of IgM and IgD with restricted kappa light chain, in most cases (59,7%). This observation highlights the importance of immunophenotyping for correct diagnosis of chronic lymphoproliferative syndromes and the panel of MoAb used was sufficient for diagnostic confirmation of B-CLL

Avaliação dos marcadores celulares por citometria de fluxo nos portadores de leucemia mieloide aguda atendidos no Hemocentro do Rio Grande do Norte-Hemonorte

The acute myeloid leukemia (AML) is a disease in which malignant myeloblasts expand, build up and suppress normal hematopoietic activity would represent a major diagnostic challenge. With the advent of immunophenotyping by flow cytometry, the diagnosis of these tumors have become more faithful, facilitating the treatment and monitoring of patients. The objectives of this study: diagnosis and classification of AML based on immunophenotyping by flow cytometry with a panel of AcMo specific for acute leukemias, set the frequency of AML in samples from patients with acute leukemias sent to the Department of Hematology Blood Center of Rio Grande do Norte - HEMONORTE, establish standards of antigen expression for different subtypes of acute leukemia and its correlation with the newly diagnosed cases refractory to treatment and recurrence of the disease, standardization of methods for detection and labeling of surface antigens by flow cytometry and intracytoplasmic flow, and observe the frequency of acute leukemia with aberrant phenotypes rare. During the study, 351 were diagnosed acute leukemia, and 179 (51%) classified as AML and 172 (49%) and ALL, which were excluded from the present work. Of the 179 AML, 92 (51.4%) were female and 87 (48.6%) were male, with ages ranging from 3 to 95 years of ag, with higher incidence in individuals in the age group of 41 to 65. Splenomegaly was the clinical finding more present, a total of 147 cases (82.1%), followed by hepatomegaly present in 132 cases (73.7%). The hemorrhagic events were observed in 55 cases (30.7%). Lymphadenopathy in turn was detected in 20 of 179 cases (11.2%). In order to classify subtypes of AML, we used a large panel of monoclonal antibodies, obtaining the following results: AML M0, 02 (1.1%) AML M1, 40 (22.3) AML M2, 60 (33.5) AML M3, 22 (12.3%) AML M4, 10 (5.6) AML M5, 13 (7.3%) AML M6 06 (3.4%) and AML M7 01 (0.6%). We observed some cases with aberrant expression of some antigens such as CD7, CD4, CD19, CD3, CD5 and TdT, CD 7 was present in 30 (16.8%), CD4 in 5 (2.8%), the CD 3 in 5 (2.8%), the CD19 in 3 (1.7%), the CD5 in 3 (1.7%) and TDT was in 7 (3.9%) cases of AML .the CD8 and CD79a was present in only a 1 case.

Investigação das alterações citogenéticas em pacientes pediátricos com leucemia linfóide aguda do Rio Grande do Norte

Leukemia is a heterogeneous group of hematologic malignancies that result from partial or total transformation of the blast cells. The Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in childhood, especially in male, Caucasian children younger than 14 years. Several criteria are adopted to classify ALL, including the cell morphology, cytochemistry, immunophenotyping and cytogenetic analysis. Cytogenetic studies allow a more detailed analysis to detect chromosomal abnormalities of leukemic cells. These modifications will determine the diagnosis, classification, stage characterization, remission assessment and prognosis. In this study were evaluated 30 patients, aged from four months to seventeen years, of both sexes and various ethnicities. The age distribution showed that 67% of patients had between one and ten years (with mean age of XX years old), the most prevalent ethnic was Caucasian (50%) and 57% were males. According to immunophenotype, 93% of patients had B-cells progenitor ALL and 7% early lineage T. Considering the total studied population, the most frequent medical findings were lymphadenopathy (37%), hepatomegaly (77%) and splenomegaly (70%), where one patient could present more than one of these medical findings. Regarding the CBC, the majority of patients had hemoglobin below 10 g / dl (73%), leukocyte count less than 10.000/μL (60%) and platelet count below 150.000/μL (83%). Chromosomal abnormalities were observed in 64% of all patients, where hyperdiploidy was the most common numerical change (67%), followed by hypodiploid (33%). All these data are in agreement with the literature. Moreover, complexes structural and/or number changes not yet described in literature were observed, which indicated poor prognosis. Finally, we concluded that this study demonstrated the importance of cytogenetic study in the diagnosis and identification of prognostic factors in pediatric patients with ALL in Rio Grande do Norte. The results obtained in this study are extremely useful and emphasizes that surveys of this nature must be conducted more frequently in our state

High-resolution electrocardiography in the diagnosis of arrhythmogenic right ventricular cardiomyopathy in Boxer dogs

Com o objetivo de avaliar a importância da eletrocardiografia de alta resolução no diagnóstico da cardiomiopatia arritmogênica do ventrículo direito do Boxer, 20 cães sem evidências de doença cardíaca estrutural à avaliação ecodopplercardiográfica foram agrupados de acordo com a frequência de arritmias ventriculares, avaliadas pela eletrocardiografia ambulatorial de 24 horas, e submetidos ao exame eletrocardiográfico de alta resolução. Duração do complexo QRS filtrado, duração dos sinais de baixa amplitude (menor que 40µV) dos últimos 40 milissegundos do complexo QRS e raiz quadrada média da voltagem ao quadrado dos últimos 40 milissegundos do complexo QRS (RMS40) foram as variáveis avaliadas. Não foram observadas diferenças significativas entre os grupos em relação às variáveis estudadas. Sendo assim, os resultados do presente estudo sugerem que a eletrocardiografia de alta resolução não é uma ferramenta útil no auxílio diagnóstico da cardiomiopatia arritmogênica do ventrículo direito dos cães da raça Boxer que não apresentam alterações miocárdicas evidentes ou disfunção sistólica.

A comparison of two agar gel immunodiffusion methods and a complement fixation test for serologic diagnosis of Brucella ovis infection in experimentally infected rams

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular diagnosis of Anaplasmataceae organisms in dogs with clinical and microscopical signs of ehrlichiosis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis

O objetivo deste estudo foi aperfeiçoar um ensaio de PCR que amplificasse um fragmento de 843 pares de bases do gene p28 da Ehrlichia canis e compará-lo com outros dois métodos de PCR utilizados para amplificar partes do gene 16S rRNA e dsb do gênero Ehrlichia. Amostras sanguíneas foram colhidas de cães com diagnóstico clínico de erliquiose. A amplificação do gene p28 pela PCR produziu um fragmento de 843pb e esse ensaio permitiu a detecção do DNA de um parasita dentre 1 bilhão de células. Todas as amostras positivas detectadas pela PCR baseada no gene p28 foram também positivas pela nested PCR para detecção do gene 16S rRNA e também pela PCR dsb. Dentre as amostras negativas para a PCR p28, 55,3% foram co-negativas, mas 27,6% foram positivas pela PCR baseada nos genes 16S rRNA e dsb. A PCR p28 parece ser um teste útil para detecção molecular de E. canis, entretanto otimizações na sensibilidade nesta PCR são necessárias, para que esta técnica se torne uma importante alternativa no diagnóstico da erliquiose canina.

Compositional nutrient diagnosis of corn using the Mahalanobis distance as nutrient imbalance index

Parent, L. E., Natale, W. and Ziadi, N. 2009. Compositional nutrient diagnosis of corn using the Mahalanobis distance as nutrient imbalance index. Can. J. Soil Sci. 89: 383-390. Compositional nutrient diagnosis (CND) provides a plant nutrient imbalance index (CND - r(2)) with assumed chi(2) distribution. The Mahalanobis distance D(2), which detects outliers in compositional data sets, also has a chi(2) distribution. The objective of this paper was to compare D(2) and CND - r(2) nutrient imbalance indexes in corn (Zea mays L.). We measured grain yield as well as N, P, K, Ca, Mg, Cu, Fe, Mn, and Zn concentrations in the ear leaf at silk stage for 210 calibration sites in the St. Lawrence Lowlands [2300-2700 corn thermal units (CTU)] as well as 30 phosphorus (2300-2700 CTU; 10 sites) and 10 nitrogen (1900-2100 CTU; one site) replicated fertilizer treatments for validation. We derived CND norms as mean, standard deviation, and the inverse covariance matrix of centred log ratios (clr) for high yielding specimens (>= 9.0 Mg grain ha(-1) at 150 g H(2)O kg(-1) moisture content) in the 2300-2700 CTU zone. Using chi(2) = 17 (P < 0.05) with nine degrees of freedom (i.e., nine nutrients) as a rejection criterion for outliers and a yield threshold of 8.6 Mg ha(-1) after Cate-Nelson partitioning between low- and high-yielders in the P validation data set, D(2) misclassified two specimens compared with nine for CND -r(2). The D(2) classification was not significantly different from a chi(2) classification (P > 0.05), but the CND - r(2) classification differed significantly from chi(2) or D(2) (P < 0.001). A threshold value for nutrient imbalance could thus be derived probabilistically for conducting D(2) diagnosis, while the CND - r(2) nutrient imbalance threshold must be calibrated using fertilizer trials. In the proposed CND - D(2) procedure, D(2) is first computed to classify the specimen as possible outlier. Thereafter, nutrient indices are ranked in their order of limitation. The D(2) norms appeared less effective in the 1900-2100 CTU zone.

Diagnosis of Smith-Lemli-Opitz syndrome by ultraviolet spectrophotometry

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder due to an inborn error of cholesterol metabolism, characterized by congenital malformations, dysmorphism of multiple organs, mental retardation and delayed neuropsychomotor development resulting from cholesterol biosynthesis deficiency. A defect in 3ß-hydroxysteroid-delta7-reductase (delta7-sterol-reductase), responsible for the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol, causes an increase in 7-DHC and frequently reduces plasma cholesterol levels. The clinical diagnosis of SLOS cannot always be conclusive because of the remarkable variability of clinical expression of the disorder. Thus, confirmation by the measurement of plasma 7-DHC levels is needed. In the present study, we used a simple, fast, and selective method based on ultraviolet spectrophotometry to measure 7-DHC in order to diagnose SLOS. 7-DHC was extracted serially from 200 µl plasma with ethanol and n-hexane and the absorbance at 234 and 282 nm was determined. The method was applied to negative control plasma samples from 23 normal individuals and from 6 cases of suspected SLOS. The method was adequate and reliable and 2 SLOS cases were diagnosed.

Contribution to the laboratory diagnosis of human cryptosporidiosis

A criptosporidiose humana é uma infecção causada pelo Cryptosporidium sp, um protozoário coccídio de patogenicidade emergente e responsável por severa e prostrante diarréia aquosa em humanos, principalmente em indivíduos imunodeprimidos. O diagnóstico, feito através da utilização de esfregaços de fezes submetidos a técnicas de concentração e coloração específica pela fucsina-carbólica tem oferecido bons resultados em nosso laboratório. Tendo em vista o longo tempo despendido para a observação dos esfregaços considerando-se a pequenez das formas e o contraste da coloração, realizamos modificações no procedimento técnico da coloração ácido-resistente que resultaram em sensível melhoria das preparações: a fucsina-carbólica passou a ser deixada sobre o esfregaço por período de 3 minutos (LENNETTE et al., 1985) e procedeu-se a substituição da solução de álcool-ácido sulfúrico a 5% (HENRIKSEN & POHLENZ, 1981) por solução de ácido clorídrico a 0,5% em álcool etílico 70%, por cerca de 2 minutos (contribuição original). Estas alterações promoveram melhor remoção do excesso de fucsina-carbólica, aumentando a eficiência da etapa de descoloração e consequentemente otimizando o contraste do processo de coloração. Nestas condições, as lâminas examinadas em microscópio óptico em aumentos de 250x e 1000x tiveram a visualização dos oocistos do protozoário facilitada, sendo os mesmos observados em contraste destacado com pigmentação intensa de cor rosa-avermelhada contra coloração de fundo azulada. Vale destacar que estas modificações oferecem vantagens de rápido processamento do material e facilidade de visualização do protozoário, diminuindo o tempo de microscopia, tornando a análise das lâminas mais rápida e menos cansativa, agilizando o diagnóstico.

Immobilization of streptavidin in sol-gel films: Application on the diagnosis of hepatitis C virus

Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45 V versus Ag/AgCl using H2O2 and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease. (c) 2006 Elsevier B.V. All rights reserved.