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Projetos de Pesquisa
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LIM/03 - Laboratório de Medicina Laboratorial, Hospital das Clínicas, Faculdade de Medicina

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Agora exibindo 1 - 10 de 18
  • article 4 Citação(ões) na Scopus
    Recommendations for quality assurance in multiparametric flow cytometry: first consensus of the Brazilian Group of Flow Cytometry (GBCFLUX)
    (2015) CORREIA, Rodolfo P.; BORTOLUCCI, Ana Carolina A.; LOPES, Annelise C. W.; SANDES, Alex F.; AZAMBUJA, Ana Paula de; VIANA, Marta A.; SALES, Maria M.; YAMAMOTO, Mihoko; BACAL, Nydia S.
    ABSTRACT: The Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo [GBCFLUX]), founded on April 24, 2010, is composed of experts in flow cytometry (FC) area who have the common objective of contributing to technical and scientific advances in Brazilian clinical and research laboratories. Among GBCFLUX working groups, the Quality Control (QC) subcommittee is responsible for discussing data in the literature and contributes to the quality assurance of the pre-analytical, analytical and post-analytical process in FC. The QC subcommittee's actions began through meetings and lectures, in which data from the literature were reviewed and discussed with all participating members of the GBCFLUX. In a second step, it was decided to draw up a text of technical and scientific consensus recommendations, informative and educative, for dissemination to all FC working groups in Brazil. To this effect, a questionnaire with objective responses was designed and sent to 35 recognized Brazilian institutions, in order to evaluate the QC profile of these institutions. Thus, the QC technical-scientific recommendations, which will be described in this updating article, are intended to ensure the process quality, technical standardization, and reproducibility of results in FC.
  • article 2 Citação(ões) na Scopus
    Cell activation state influences the modulation of HLA-DR surface expression on human monocytes/macrophages by parenteral fish oil lipid emulsion
    Abnormal surface expression of HLA-DR by leukocytes is associated with a poor prognosis in critical care patients. Critical care patients often receive total parenteral nutrition with lipid emulsion (LE). In this study we evaluated the influence of fish oil LE (FO) on human monocyte/macrophage (M phi) expression of surface HLA-DR under distinct activation states. Mononuclear leukocytes from the peripheral blood of healthy volunteers (n=18) were cultured for 24 hours without LE (control) or with 3 different concentrations (0.1, 0.25, and 0.5%) of the follow LE: a) pure FO b) FO in association (1:1-v/v) with LE composed of 50% medium-chain trygliceride and 50% soybean oil (MCTSO), and c) pure MCTSO. The leukocytes were also submitted to different cell activation states, as determinate by INF-g addition time: no INF-gamma addition, 18 hours before, or at the time of LE addition. HLA-DR expression on MO surface was evaluated by flow cytometry using specific monoclonal antibodies. In relation to controls (for 0.1%, 0.25%, and 05%: 100) FO decreased the expression of HLA-DR when added alone in simultaneously-activated M., for 0.1%: 70 (59 +/- 73); for 0.25%: 51 (48 +/- 56); and for 05%: 52.5 (50 +/- 58)1 or in association with MCTSO [in simultaneously-activated MO, for 0.1%: 50.5 (47 +/- 61); for 25%: 49 (45 +/- 52); and for 0.5%: 51 (44 54) and in previously-activated Mf, for 1.0%: 63 (44 +/- 88); for 0.25%: 70 (41 +/- 88); and for 0.5%: 59.5 (39 +/- 79)1 in culture medium (Friedman p < 0.05). In relation to controls (for 0.1%, 0.25%, and 0.5%: 100), FO did not influence the expression of these molecules on non-activated M phi [for 0.1%: 87.5(75 +/- 93); for 0.25%: 111 (98 +/- 118); and for 0.5%: 101.5 (84 +/- 113)]. Results show that parenteral FO modulates the expression of HLA-DR on human MO surface accordingly to leukocyte activation state. Further clinical studies evaluating the ideal moment of fish oil LE infusion to modulate leukocyte functions may contribute to a better understanding of its immune modulatory properties.
  • article 2 Citação(ões) na Scopus
    Massive central nervous system infiltration by CD56-positive plasma cells in multiple myeloma
    (2017) LOPES, A. C. W.; XAVIER, F. D.; BARROSO, R. de Souza; GOMES, H. R.; SALES, M. M.
  • conferenceObject
    (2012) WENGERKIEVICZ, Annelise Correa; BENTO, Laiz Cameirao; XAVIER, Flavia Dias; SANTOS, Michelle Carolina dos; KONECSNI, Cecilia Ana; SALES, Maria Mirtes
    Objectives: Clinical flow cytometric analysis of neoplastic hematolymphoid cells relies on evaluation of surface membrane and intracellular antigens expression patterns, which are related to normal cells of a particular lineage and maturational stage. A complete characterization of the studied population includes information about presence, absence and level of expression of a series of relevant antigens recognized by highly specific monoclonal antibodies (MAbs). Usually, the fluorescence intensity is considered to be directly proportional to the amount of antigen expressed in a specific population, as assessed by flow cytometry using the mean peak of channel fluorescence (MPCF), the proper use of MAbs is essential for this condition to be valid. Data show that laboratories use a wide variety of MAb dilutions, even those obtained from the same manufacturer. Ideally, the titre value should be determined for every MAb and is defined as the amount of antibody that is required to satura te the maximum number of antigen binding sites on a selected cell population, ensuring that antibodies are used efficiently yet still remain in excess. Our objective is to report a new statistical criteria adopted by our laboratory to establish optimal MAb dilution for flow cytometry analysis based on the discrimination of unstained and positively stained cells using MPCF. Methods: We have tested in our laboratory 57 MAbs from different manufacturers, from May/2010 until August/2011 (27 conjugated to FITC, 11 to PE, 4 to PerCP/PEDY647/CyQ or RPECy5 and 5 to APC). The tests were performed using bone marrow or peripheral blood samples which presented any positive population (normal or neoplastic) for the studied MAbs. The staining procedure followed manufacturers ́ protocols, with different volumes of Mabs (recommended by manufacturer followed by titrations 1:2, 1:4 and >1:4). At least 10,000 events were acquired for each sample using the flow cytometer BD FACSCalliburTM, and data was analyzed by CellQuestTM, obtaining the MPCF values for unstained and positively stained cells. The percentage difference between the MPCF (PD-MPCF) values was calculated applying this formula: (MPCF from positively stained population–MPCF from unstained population) x 100/MPCF from positively stained population. An adequate PD-MPCF must be 95% or more. Results: Among MAbs tested, 42/57 (73,68%) were likely to be titrated, 18 being titrated to 1:2, 22 to 1:4 and 2 to >1:4. Analysis according to the fluorochrome conjugated to MAb demonstrated more efficient titration for APC (3 MAbs in 5), while those conjugated with FITC showed difficulty in titration (9/27 have not shown good performance at lower concentrations than those indicated by the manufacturer and titers >1:4 were not appropriate for any samples). Six MAbs had titration disapproved by the criterion used (PD-MPCF) but were also disapproved in the concentration recommended by the manufacturer because the expected fluorescence level was dim for the population analyzed. Conclusions: We conclude that PD-MPCF is an efficient and objective instrument as quality control for validation and titration of MAbs, and that the cutoff of 95% ensures proper separation between positive and negative stained populations showing good correlation with the performance in daily routine.
  • conferenceObject
    Impact of Treatment On CD4+CTLA-4+T Cell Subset in Brazilian Patients with Classical Hodgkin Lymphoma
    (2012) SILVA, Joyce M. K.; SALES, Maria Mirtes; PENNA, Adriana M. Damasco; CHAVES, Elma Maria Chaves Maria; SILVA, Priscilla B.; ASSIS, Mariane Cristina Gennari; BAIOCCHI, Otavio Cesar Carvalho Guimaraes
    Cytotoxic T lymphocyte antigen-4 (CTLA-4) is one of the basic antigens involved in immune responses regulation associated with autoimmune diseases and cancer. Its key role in regulating the immune system has made CTLA-4 an attractive target for cancer. Augmentation of the immune response via blockade of CTLA-4 has shown an improvement in survival for patients with metastatic melanoma, which prompted the Food and Drug Administration (FDA) approval of the CTLA-4 function blocking antibody Ipilimumab for this disease. Objective: The aim of the study was to evaluate the surface expression of CTLA-4 on CD4+ T cells in peripheral blood mononuclear cells (PBMC) of patients with classical Hodgkin lymphoma (cHL) at diagnosis and post-treatment and correlate these findings with clinical and epidemiological aspects. Material and Methods: This is an open study and, so far, we included 35 patients from December 2009 to December 2011. Blood was drawn at diagnosis and post-treatment (1 to 4 months after completion of therapy). The T cell phenotype was evaluated by flow cytometry using CD3, CD4, CD8, CTLA-4 and correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. Eighteen healthy blood donors volunteers were recruited as controls. In this study, only cHL patients whose histology could be confirmed and Epstein-Barr (EBV) association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. Three patients relapsed, and blood was also drawn at this time. Results: From the 35 cHL patients, 17 were EBV related and 18 EBV non-related. The percentage of CD4+ T cells with CTLA-4 surface expression was significantly increased in patients with cHL at diagnosis compared with healthy controls (median 7.36 vs 2.73; P<0.001). Additionally, CD4+CTLA-4+ T lymphocytes significantly decreased following treatment and complete response (7.36 vs 4.53; p=0.008), with values similar to healthy controls (4.53 vs 2.73; p=0.07). Interestingly, CD4+CTLA-4+ T lymphocytes on relapse were significantly different from post-treatment values and similar to pre treatment. There was no difference on CD4+CTLA-4+ T lymphocytes in the EBV related and non-related cHL patients. Regarding patient's baseline characteristics, CD4+CTLA-4+ T lymphocytes strongly correlated with erythrocyte sedimentation rate (ESR) values (r=0.67; p=0.002). Conclusions: We showed that CD4+CTLA-4+ T lymphocytes are increased in Brazilian cHL patients at diagnosis compared with post-treatment values and healthy controls. These results suggest a role of CTLA-4 on Hodgkin lymphomagenesis, possibly negatively regulating host anti-tumor immune response. The promising immunotherapy regimen targeting CTLA-4 might be beneficial in classical Hodgkin lymphoma.
  • conferenceObject
    Quantification of Regulatory/Effector CD4(+) T Lymphocytes Pre and Post-Treatment in Patients with Classical Hodgkin Lymphoma in Brazil
    (2012) SILVA, Joyce M. K.; PENNA, Adriana M. Damasco; SALES, Maria Mirtes; CHAVES, Elma Maria; SILVA, Priscilla Brito; ASSIS, Mariane Cristina Gennari; BAIOCCHI, Otavio Cesar Carvalho Guimaraes
    Introduction: Epstein-Barr virus (EBV) can be found latently infecting Reed-Sternberg (RS) malignant cells in approximately 50% of classical Hodgkin lymphoma (cHL) patients in Brazil. EBV signaling leads to a disbalance between effector and regulatory CD4 T lymphocytes in the tumor microenvironment, promoting the immune evasion of RS malignant cells. However, little is known about these lymphocytes subpopulations in the peripheral blood of patients with cHL and how treatment can modify this regulatory/effector ratio. In this study, we analyzed the regulatory and effector CD4+ subpopulations in peripheral blood in patients with EBV related and non-related cHL and the impact of treatment on these cells. Material and Methods: This is an open multicentric study and, so far, we included 35 patients from December 2009 to December 2011. Blood was drawn at diagnosis and after completion of treatment (1 to 4 months). Eighteen healthy blood donors volunteers were recruited as controls. Quantification of regulatory and effector T lymphocytes was done by flow cytometry using CD3, CD8, CD4, CD25, Foxp3, GITR, CD127 and interleukin-17 (IL17) antibodies and correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. In this study, only cHL patients whose histology could be confirmed and EBV association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. Results: From the 35 cHL patients, 17 were EBV related and 18 EBV non-related. The percentage of CD4+CD25highFoxP3+ and CD4+GITR+ at diagnosis was significantly different from healthy controls (median 1.04 vs 0.26, p=0.02; 4.2 vs 2.2, p=0.003; respectively). CD4+CD127+ T lymphocytes were not different from controls (p=0.3). Additionally, CD4+ T lymphocytes with effector phenotype (CD4+IL17+) were significantly increased in cHL patients compared with controls (0.42 vs 0.13, p<0.001). When we compared pre-treatment values of regulatory and effector CD4+ T lymphocytes with post-treatment values, we did not find any statistical difference. Interestingly, post-treatment values were not statistically different from healthy controls. There was no difference on regulatory and effector CD4+ T lymphocytes in the EBV related and non-related cHL patients. Regarding patient's baseline characteristics, patients with advanced disease and B symptoms presented with increased CD4+CD25highFoxP3+ and CD4+GITR+ T lymphocytes (p=0.03 and p=0.01, respectively). Conclusions: Our results demonstrate that patients with cHL presented with increased CD4+CD25highFoxP3+, CD4+GITR+ and CD4+IL17+ at diagnosis compared with healthy controls. Also, treatment had no impact on these CD4+ T lymphocytes populations. Probably, the moment blood was drawn after completion of therapy could have influenced our results as we know that immunological reconstitution in patients with cHL may take several months. Further studies investigating these CD4+ T lymphocytes subpopulations together with functional assays will contribute not only to our understanding on the pathogenesis of cHL but also to the development of therapeutic strategies designed to manipulate regulatory activity. Given that the incidence of EBV-related cHL, disease presentation and severity are different in developing countries than in developed ones, we emphasize the importance of this ongoing Brazilian multicentric project.
  • article 49 Citação(ões) na Scopus
    Proteína C reativa: aplicações clínicas e propostas para utilização racional
    (2013) AGUIAR, Francisco J. B.; FERREIRA-JUNIOR, Mario; SALES, Maria M.; CRUZ-NETO, Luiz M.; FONSECA, Luiz A. M.; SUMITA, Nairo M.; DUARTE, Nilo J. C.; LICHTENSTEIN, Arnaldo; DUARTE, Alberto J. S.
    C-reactive protein (CRP) is an acute-phase protein whose requests have been growing exponentially in several countries, including Brazil. In this study, the use of CRP in several clinical situations was reviewed by a group of physicians comprised by specialists in internal medicine, medical emergencies, intensive care, screening, and laboratory medicine, aiming to analyze the applicable literature and to propose guidelines for a more rational use of this laboratory test. The result was the creation of flowcharts guiding CRP request, adjusted to four different healthcare environments, namely, intensive care units, emergency room, wards; and outpatient clinics. These flowcharts, as well as a more detailed discussion on several clinical recommendations for the test, are presented in this study.
  • article 33 Citação(ões) na Scopus
    Prolonged suppression of monocytic human leukocyte antigen-DR expression correlates with mortality in pediatric septic patients in a pediatric tertiary Intensive Care Unit
    (2016) MANZOLI, Talita Freitas; TROSTER, Eduardo Juan; FERRANTI, Juliana Ferreira; SALES, Maria Mirtes
    Introduction: Inimunoparalysis is a syndrome with no clinical symptoms that occurs in some septic patients. Monocytic human leukocyte antigen-DR (mHLA-DR) expression has been used to identify patients in immunoparalysis and prolonged periods of reduced mHLA-DR expression have been con-elated with a poor prognosis in sepsis. However, there is a lack of studies investigating mHLA-DR expression in pediatric septic patients. Aim: To determine if mHLA-DR expression correlates with mortality in pediatric septic patients using the QuantiBRITE Anti HLA-DR/Anti-Monocyte,a Bechton Dickinson novel reagent that standardizes flow cytome try values. Methods: We determined mHLA-DR expression in 30 patients with severe sepsis or septic shock admitted to the pediatric intensive care unit at Hospital das Clinicas da Faculdade de Medicina (la Universidade de Sao Paulo, Sao Paulo, Brazil, between January 2013 and February 2015. mHIA-DR expression was quantified between days 3 to 5 and 5 to 7 after the onset of sepsis and the AmHLA-DR (mHLA-DR2 mHLA-DR1) was calculated. We also measured mHLA-DR levels in 21 healthy control patients. Results: Mean mHLA-DR expression was significantly lower in septic patients than in controls (P.0001). Mortality was 46% in patients with negative AHLA-DR or <1000 mAblcell and 7% in patients with positive AHLA-DR or >1000 mAID/cell. Mean AmHLA-DR levels were significantly different between survivors and non survivors (P.023). Conclusion: AHLA-DR correlates with mortality in pediatric patients with septic shock or severe sepsis. This is the first study to have used the QuantiBRITE Anti HLA-DR/Anti-Monocyte reagent- to quantify monocyte HLA-DR expression in pediatric septic patients.
  • bookPart
    Citometria de fluxo
    (2016) SALES, Maria Mirtes
  • article 3 Citação(ões) na Scopus
    Rational use of blood calcium determinations
    (2014) FERREIRA-JUNIOR, Mario; LICHTENSTEIN, Arnaldo; SALES, Maria Mirtes; TANIGUCHI, Leandro Utino; AGUIARI, Francisco Jose Bueno de; FONSECA, Luiz Augusto Marcondes; SUMITA, Nairo Massakazu; DUARTE, Alberto Jose da Silva
    CONTEXT AND OBJECTIVE: This study was motivated by the recent excessive increase in requests for blood calcium determinations and laboratory tests in general, in the Hospital das Clinicas complex of Faculdade de Medicina, Universidade de Sao Paulo (HCFMUSP). Its aim was to suggest rules for the determination of total and ionized calcium in our intensive care units, emergency department, wards and outpatient services, thus contributing towards improving the quality of medical care and achieving more appropriate use of human and financial resources. DESIGN AND SETTING: Critical analysis on clinical and laboratory data and the pertinent scientific literature, conducted by the study group for rational clinical laboratory use, which is part of the Central Laboratory Division, HCFMUSP. METHODS: The study group reviewed scientific publications, statistics and clinical and laboratory data concerning requests for total and ionized calcium determinations in the settings of intensive care units, emergency department, wards and outpatient services. RESULTS: From this critical analysis, clinical decision flow diagrams aimed at providing guidance for ordering these tests were constructed. CONCLUSIONS: Use of the proposed flow diagrams may help to limit the numbers of inappropriate requests for ionized and total calcium determinations, with consequent reductions in the number of tests, risks to patients and unnecessary costs.