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1.
Rev. cuba. hematol. inmunol. hemoter ; 38(2): e1646, abr.-jun. 2022. tab, graf
Article in Spanish | LILACS, CUMED | ID: biblio-1408460

ABSTRACT

Introducción: Los cambios en el inmunofenotipo de los linfocitos en los pacientes con linfoma no Hodgkin están asociados con el pronóstico y las respuestas terapéuticas. Sin embargo, no se ha establecido sistemáticamente la asociación con la enfermedad y por tanto su contribución al diagnóstico. Objetivo: Evaluar la asociación del inmunofenotipo linfocitario en sangre periférica con la presencia del linfoma no Hodgkin. Métodos: Se analizaron 31 muestras de sangre periférica de pacientes con diagnóstico confirmado de linfoma no Hodgkin y de 68 individuos sanos como controles, durante el período de 2018 a 2020. Se empleó la citometría de flujo multiparamétrica para el inmunofenotipado. Se calculó el área bajo la curva y el índice de Youden para establecer puntos de corte en los porcentajes linfocitarios. La asociación de los cambios inmunofenotípicos con el linfoma no Hodgkin, se realizó mediante cálculos de Odd ratio. Resultados: El aumento de linfocitos TCD8+ y NKCD56opaco se asoció significativamente con la presencia de linfoma no Hodgkin (OR= 3,4 y 2,9; respectivamente). Por el contrario, la disminución de linfocitos TCD4+, T doble positivo, T doble negativo y NKCD56brillante también se asoció con la existencia de linfoma no Hodgkin (OR= 23,0; 10,7; 6,9 y 15,8; respectivamente). Además, la disminución del índice CD4/CD8 también fue asociada con la enfermedad. Conclusiones: Los cambios encontrados en los inmunofenotipos linfocitarios se asociaron de forma significativa con la presencia del linfoma no Hodgkin, lo cual representa una expresión sistémica de la enfermedad y sugiere su valor diagnóstico(AU)


Introduction: Lymphocyte immunophenotype changes in non-Hodgkin lymphoma patients are associated with prognosis and therapeutic responses. However, its association with the disease has not been systematically established. Therefor its contribution to the diagnosis process. Objective: To assess the association of lymphocyte immunophenotype in peripheral blood with the presence of non-Hodgkin lymphoma. Methods: 31 peripheral blood samples were analyzed from patients with a confirmed diagnosis of non-Hodgkin lymphoma and from 68 healthy individuals as controls, during the period 2018 to 2020. Multiparametric flow cytometry was used for immunophenotyping. The area under the curve and the Youden index were calculated to establish cut-off points in lymphocyte percentages. The association of immunophenotypic changes with non-Hodgkin's lymphoma was made using Odd ratio calculations. Results: The increase in TCD8+ and NKCD56dim lymphocytes from peripheral blood was significantly associated with the presence of non-Hodgkin lymphoma (OR= 3.4 and 2.9, respectively). Oppositely, the decrease in TCD4+, double positive T, double negative T and NKCD56bright lymphocytes was associated with the existence of non-Hodgkin lymphoma (OR= 23.0, 10.7, 6.9 and 15.8, respectively). Therefore, the decrease in the CD4/CD8 rate was also associated with the disease. Conclusion: The changes found in these lymphocytic immunophenotypes were significantly associated with the presence of non-Hodgkin lymphoma, which represents a systemic expression of the disease and suggests its diagnostic value(AU)


Subject(s)
Humans , Lymphoma, Non-Hodgkin , CD4 Antigens , Immunophenotyping/methods , CD8 Antigens , Flow Cytometry/methods
2.
São Paulo; s.n; s.n; 2022. 141 p. graf, tab.
Thesis in Portuguese | LILACS | ID: biblio-1379329

ABSTRACT

Vários estudos epidemiológicos estabelecem correlação positiva entre os níveis de ácido úrico sérico e o aumento do risco para doenças cardiovasculares. Fatores dietéticos e socioeconômicos, além da presença de comorbidades estão diretamente associados aos níveis séricos de ácido úrico. Países desenvolvidos apresentam maior incidência e prevalência da gota e alguns grupos étnicos são particularmente susceptíveis à hiperuricemia. Cristais de ácido úrico são descritos por iniciar e perpetuar resposta inflamatória, e sinalizar um padrão de resposta molecular associado ao dano (DAMP), permitindo a diferenciação de macrófagos para perfis pró-inflamatórios. Por outro lado, os efeitos do ácido úrico em sua forma solúvel ainda carecem de estudos. Macrófagos derivados de precursores monocíticos apresentam diferenciação específica e respondem a um conjunto de fatores extrínsecos, resultando em perfis distintos, um fenômeno conhecido como polarização. Assim, os macrófagos podem ser classicamente ativados para uma resposta Th1 (T helper 1) e polarizados a um perfil pró- inflamatório (M1, resposta Th1) ou a um perfil alternativo e oposto, um perfil de resolução da inflamação (M2, resposta Th2, T helper 2). Nesse sentindo, buscamos analisar os efeitos do ácido úrico solúvel sobre vias de modulação da polarização fenotípica de macrófagos e modificação redox. Utilizamos a linhagem monocítica humana THP-1, a qual foi diferenciada em macrófagossímile por acetato miristato de forbol (PMA; 5 ng.mL-1) por 48 h, seguidas da incubação com ácido úrico em meio ausente de tióis e soro fetal bovino por 8h ou 24h (0-1000 µM). A expressão de fatores de transcrição e marcadores de polarização foi realizada através de citometria de fluxo, western-blotting e por microscopia de fluorescência com alto conteúdo de imagens (HCI). Em concentrações fisiológicas, verificamos que o ácido úrico solúvel regulou positivamente a frequência de células para receptor manose CD206, um marcador clássico de perfil alternativo/M2 e regulou negativamente a expressão óxido nítrico sintase induzível (iNOS), um marcador M1, sugerindo inicialmente uma modulação para o perfil de polarização M2. Além disso, as proteínas redoxsensíveis, heme oxigenase-1 (HO-1) e tiorredoxina (Trx) tiveram sua expressão reduzida e aumentada, respectivamente, pelo tratamento com ácido úrico. Os fatores de transcrição Nrf2 e STAT3 tiveram regulação negativa após a exposição ao ácido úrico solúvel. Os resultados apresentados nesta tese sugerem uma função do urato no priming de macrófagos através da alteração da polarização destas células


Several epidemiological studies have established a positive correlation between high serum uric acid levels and increased risk for cardiovascular diseases. Developed countries have a higher incidence and prevalence of gout and some ethnic groups are particularly susceptible to hyperuricemia. Although hyperuricemia is a prevalent condition, it has still controversy biological consequences. Uric acid crystals are described as capable of initiating and perpetuating inflammatory responses, by activating the damage-associated molecular response pattern (DAMP) cascade, allowing macrophage differentiation to inflammatory profiles. In spite of that, biological response to soluble uric acid are not completely understood. Monocyte-derived macrophages respond to a set of extrinsic factors that result in different profiles and can be polarized to a proinflammatory (M1) or anti-inflammatory (M2) profile. In this thesis, we analyzed the effects of soluble uric acid on redox-modulated pathways and the phenotypic polarization of macrophages. We used human monocytic THP-1 cell line, differentiated into macrophage by phorbol myristate acetate (PMA; 5 ng.mL-1) for 48 h. After differentiation, cells were incubated with soluble uric acid in medium without thiols and fetal bovine serum for 8 h and 24 h (0-1000 µM). The expression of transcription factors and polarization markers were assessed by flow cytometry, western-blotting and fluorescence microscopy with high content imaging (HCI). At physiological concentrations, soluble uric acid positively regulated the frequency of cells for mannose receptor CD206, a classic marker of the anti-inflammatory M2 profile and negatively regulated the inducible nitric oxide synthase (iNOS) expression, a proinflammatory M1 marker, suggesting that the soluble uric acid changes the polarization profile to M2 profile. In addition, the redox-sensitive proteins heme oxygenase-1 (HO-1) and thioredoxin (Trx) had their expression decreased and increased, respectively, after exposure to urate. STAT3 and Nrf2 transcription factors were downregulated upon soluble uric acid exposure. The results presented in this thesis suggest a role of uric acid in macrophage priming through the alteration of cell polarization


Subject(s)
Uric Acid/analysis , THP-1 Cells/classification , THP-1 Cells/chemistry , Inflammation/classification , Macrophages/chemistry , Sulfhydryl Compounds/agonists , Cardiovascular Diseases , Epidemiologic Studies , Nitric Oxide Synthase Type II/antagonists & inhibitors , Flow Cytometry/methods , Microscopy, Fluorescence/methods
3.
São Paulo; s.n; s.n; 2022. 129 p. tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-1392257

ABSTRACT

O objetivo deste estudo foi desenvolver uma formulação de bebida láctea bubalina probiótica adicionada de polpa de morango, comparando os efeitos do uso do leite de búfala e de vaca na elaboração dos produtos e verificando a possibilidade de suplementação com triptofano nos produtos lácteos probióticos. Como primeira etapa do trabalho, bebidas lácteas probióticas foram elaboradas a partir de leite bubalino e bovino, fermentadas com Streptococcus thermophilus TA040, Lactobacillus bulgaricus LB340 e Lactobacillus acidophilus La5, e formuladas com 0, 25 e 50% de soro em sua formulação. As bebidas foram avaliadas quanto à cinética de fermentação das culturas láticas utilizadas, ao teor de proteína, gordura e sólidos totais não gordurosos, pós-acidificação, viabilidade das culturas fermentadoras e sua capacidade de sobrevivência ao estresse gastrointestinal in vitro. As bebidas lácteas bubalinas apresentaram resultados superiores as bebidas bovinas. O uso do leite de búfala na elaboração das bebidas lácteas promoveu benefícios quanto as culturas láticas presentes nos produtos, exercendo efeito protetivo e influindo na preservação da viabilidade das bactérias ao longo do armazenamento refrigerado e durante a simulação do estresse gastrointestinal in vitro. As bebidas lácteas elaboradas com 25% apresentaram os resultados mais próximos aos obtidos pelos produtos controle, sem adição de soro, sendo selecionadas para a segunda parte do estudo. Nesta etapa, as formulações de bebida láctea com 25% de soro, foram acrescidas de um preparado com polpa de morango e bebidas sem adição da fruta, utilizadas como controle. As bebidas lácteas bubalinas frutadas, apresentaram menor teor de gordura e melhores características reológicas, com maior viscosidade e consistência do que os produtos controle, sem afetar a pós-acidificação, o perfil de ácido graxo, assim como, a viabilidade e a resistência às condições de estresse gastrointestinal in vitro das culturas fermentadoras. A avaliação da possibilidade de suplementar lácteos probióticos com triptofano foi realizada em conjunto com a Universidade de Milão. Para isso, iogurtes probióticos receberam adição de triptofano antes ou após a fermentação, sendo avaliados com relação ao perfil de pós-acidificação, quantidade de triptofano nos produtos, número de células viáveis por plaqueamento e citometria de fluxo ao longo do armazenamento a 25° e 4°C. Complementarmente, a influência da presença do triptofano no crescimento e produção de compostos antimicrobianos pelas culturas láticas, também foi avaliada. A adição de triptofano após a fermentação dos iogurtes, que foram armazenados sob refrigeração (4°C), além de não afetar a pós-acidificação dos produtos, apresentou benefícios quanto a viabilidade L. acidophilus, redução do dano e aumento do número de células vivas, promovendo teor maior do aminoácido nos iogurtes. A presença do triptofano nos meios de cultivo, também influenciou de forma positiva o crescimento de S. thermophilus e L. acidophilus, melhorando o desenvolvimento das bactérias durante a fermentação e influindo em uma maior atividade antilistérica por parte do S. thermophilus. Diante da influência positiva da aplicação do leite de búfala na elaboração das bebidas lácteas, assim como, a adição do triptofano em iogurtes probióticos, a suplementação do aminoácido em bebidas lácteas bubalinas frutadas permitiria a obtenção de um produto funcional, onde seus benefícios estariam relacionados tanto ao consumo do probiótico presente no produto quanto a complementação de triptofano na dieta do consumidor


The aim of this study was to develop a formulation of probiotic buffalo dairy beverage added with strawberry pulp, comparing the effects of using buffalo and cow's milk in the preparation of products and verifying the possibility of tryptophan supplementation in probiotic dairy products. As a first stage of the work, probiotic dairy beverages were made from buffalo and bovine milk, fermented with Streptococcus thermophiles TA040, Lactobacillus bulgaricus LB340 and Lactobacillus acidophilus La5, and formulated with 0, 25 and 50% whey in their formulation. The beverages were evaluated for the fermentation kinetics of the used lactic cultures, the levels of protein, fat and total no fat solids, post-acidification, fermenting cultures viability and their ability to survive gastrointestinal stress in vitro. Buffalo milk use in dairy beverages production promoted benefits regarding the lactic cultures present in the products, exerting a protective effect and influencing the viability preservation of bacteria during the cold storage and simulation of gastrointestinal stress in vitro. Dairy beverages made with 25% whey addition showed results similar to those obtained by the control products, without whey addition, being selected for the second part of the study. In this part, the dairy beverages formulations with 25% whey, were added with a preparation were added with a strawberry pulp preparation and dairy beverages without added fruit, used as a control. Fruity bubaline dairy beverages had lower fat content and better rheological characteristics, with higher viscosity and consistency than control products, without affecting post-acidification, fatty acid profile, as well as viability and resistance to in vitro gastrointestinal condition of fermented cultures. The possibility of supplementing probiotic dairy products with tryptophan was evaluated in partnership with the University of Milan. For this, probiotic yogurts received the addition of tryptophan before or after fermentation, being evaluated in relation to the post-acidification profile, tryptophan amount in the products, viable cell number per plating and flow cytometry during storage at 25°C and 4°C. In addition, the influence of the tryptophan presence on the growth and production of antimicrobial compounds by lactic cultures was also evaluated. The addition of tryptophan after the yogurt fermentation, which were stored under refrigeration (4°C), in addition to not affecting the post-acidification of the products, showed benefits to the viability of L. acidophilus, reduced the damage and increased the number of cells promoting higher amino acid content in yogurts. Tryptophan presence in the culture media also positively influenced the growth of S. thermophiles and L. acidophilus, improving the development of bacteria during fermentation and influencing better antilisteric activity in the part of S. thermophiles. In view of the buffalo milk positive influence observed after the application in dairy beverage preparation, as well as the addition of tryptophan in probiotic yoghurts, amino acid supplementation in fruity buffalo dairy beverages would allow to obtain a functional product, where its benefits would be related both to the consumption of the probiotic present in the product as to the supplementation of tryptophan in the consumer's diet


Subject(s)
Beverages/adverse effects , Milk/adverse effects , Tryptophan/classification , Yogurt , In Vitro Techniques/methods , Buffaloes , Cell Count/instrumentation , Chemistry, Pharmaceutical , Probiotics/classification , Streptococcus thermophilus/metabolism , Lactobacillus delbrueckii/metabolism , Growth and Development , Flow Cytometry/methods , Whey/adverse effects , Fruit , Amino Acids/antagonists & inhibitors , Lactobacillus acidophilus/metabolism
4.
Braz. J. Pharm. Sci. (Online) ; 58: e19400, 2022. tab, graf
Article in English | LILACS | ID: biblio-1403750

ABSTRACT

Abstract This study highlights the cytotoxic effect of three L. casei strains on colorectal cell lines in invitro conditions. Different concentrations of live, heat killed (HK) and cell free supernatant (CFS) of three L.casei strains were subjected to CaCo2 and MRC5 cell lines. The viability of the treated and untreated cells was determined after 72 hrs by MTT assay, and IC50 estimated. Apoptosis was evaluated by Annexin V-propidium iodide method using flow cytometry. The live, HK and CFS of the L. casei strains showed cytotoxic effects on colorectal cell lines with significant differences. The cytotoxicity effects of live cells on CaCo2 cells were significantly higher (p˂0.01) than the HK cells. A dose dependent response was observed, as higher concentrations resulted in enhanced cytotoxicity effects. Live L.casei 1296-2cells inhibited 91% of CaCo2 cell growth, with IC50 of less than 108 cfu/ml. MRS medium and concentrations of CFS at above 20% v/v, were cytotoxic to the normal cell lines. Flow cytometry analyses of L. casei 1296-2 indicated that cytotoxicity effects on CaCo2 cells is related to apoptotic induction. Invitro studies indicate that Live and CFS of L. casei 1296-2 might be promising candidate for the control of colorectal cancers


Subject(s)
Propidium/analysis , Colonic Neoplasms/pathology , Probiotics/analysis , Lactobacillus casei/metabolism , Colorectal Neoplasms , Cells/immunology , Apoptosis , Inhibitory Concentration 50 , Flow Cytometry/methods
5.
Article in Chinese | WPRIM | ID: wpr-939707

ABSTRACT

OBJECTIVE@#To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample.@*METHODS@#Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope.@*RESULTS@#It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV+ cells from cell lines and blood samples of patients were identified successfully.@*CONCLUSION@#A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.


Subject(s)
Epstein-Barr Virus Infections , Flow Cytometry/methods , Herpesvirus 4, Human , Humans , In Situ Hybridization, Fluorescence/methods , Lymphocyte Subsets
6.
Article in Chinese | WPRIM | ID: wpr-928717

ABSTRACT

OBJECTIVE@#To establish 10-color fluorescent antibody combination panels for the detection of minimal residual disease (MRD) of acute myeloid leukemia (AML) in our laboratory and discuss the value of clinical application.@*METHODS@#According to the antigen expression characteristics of leukemia cells of incipient AML patients, MRD in bone marrow were detected by multiparameter flow cytometry, and the test results were compared with both bone marrow cell morphology and PCR results, then 10-color fluorescent antibody combination panels in our lab for MRD detection was determined.@*RESULTS@#The immunophenotypic characteristics of 392 incipient patients with AML in the First Affiliated Hospital of Zhengzhou University were analyzed, among them 357 (91.07%) cases showed abnormal immunophenotypes, which mainly included cross-lineage expression, cross-stage expression, deficiency of antigen expression or abnormal antigen intensity and other abnormal expression. The 10-color fluorescent antibody combination panels established according to abnormal immunophenotypic characteristics of leukemia cells were applied for detecting MRD in 156 patients with AML, the positive rate (43.6%) was higher than 26.8% of morphology, and the results were highly consistent with PCR detection results (96.49%), moreover, the recurrence rate of MRD positive patients (86.96%) was significantly higher than 5.75% of MRD negative patients. Therefore, this method could truly reflect the load of leukemia cells and prompt change of disease condition.@*CONCLUSION@#Multiparameter flow cytometry can detect various abnormal immunophenotypes of AML. The 10-color fluorescent antibody combination panels in our lab based on the characteristics of antigens expression in leukemia cells can well detect MRD of leukemia cells, so as to predict relapse and provide basis for clinical treatment.


Subject(s)
Bone Marrow , Flow Cytometry/methods , Humans , Immunophenotyping , Leukemia, Myeloid, Acute/diagnosis , Neoplasm, Residual/diagnosis
7.
Braz. J. Pharm. Sci. (Online) ; 58: e19692, 2022. graf
Article in English | LILACS | ID: biblio-1384014

ABSTRACT

Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.


Subject(s)
Tissue Plasminogen Activator , Process Optimization , Flow Cytometry/methods , Fluorescence , Cell Count/instrumentation , Clone Cells/classification , Plasminogen Activator Inhibitor 1/adverse effects , Green Fluorescent Proteins
8.
Rev. cuba. hematol. inmunol. hemoter ; 37(3): e1428, 2021. tab
Article in Spanish | LILACS, CUMED | ID: biblio-1341401

ABSTRACT

Introducción: La enfermedad mínima residual es la permanencia de células leucémicas residuales en niveles subclínicos luego de la remisión de la enfermedad. Esta condición incrementa el riesgo de recaída y mortalidad. Objetivo: Caracterizar factores clínicos y moleculares de pacientes con leucemias agudas y enfermedad mínima residual detectada por citometría de flujo en una institución de alta complejidad de la ciudad de Medellín, Colombia durante los años 2015 - 2017. Metodología: Este es un estudio descriptivo retrospectivo, que incluyó pacientes con leucemia diagnosticada por citometría de flujo. Se realizó un muestreo no probabilístico de casos consecutivos. La información recolectada fue digitada en una base de datos en Excel, y el análisis se realizó a través del programa IBM SPSS Versión 24, empleando según la naturaleza de cada variable frecuencias absolutas y relativas, promedio y desviación estándar o mediana y rangos intercuartílicos según su distribución. Resultados: Se incluyó un total de 60 pacientes con predominio del sexo masculino 63,3 por ciento (38). El diagnóstico más frecuente fue la leucemia linfoide 78,3 por ciento (47). Del total de pacientes incluidos, 36,6 por ciento (22) fue positivo para enfermedad mínima residual; 28,3 por ciento recibió trasplante de médula ósea y el 10 por ciento (6) presentó compromiso de líquido cefalorraquídeo. En la segunda citometría en pacientes con enfermedad mínima residual, 90,9 por ciento (20) expresaba CD45+. El 31,8 por ciento (7) de los pacientes con enfermedad mínima residual presentó recaída. Conclusión: La enfermedad mínima residual es una condición frecuente en pacientes con leucemias agudas que requiere seguimiento y constituye un factor pronóstico relevante(AU)


Introduction: The minimal residual disease is the permanence of residual leukemic cells at subclinical levels after remission of the disease. This condition increases the risk of relapse and mortality. Objective: To characterize the clinical and molecular factors of patients with acute leukemias and minimal residual disease detected by flow cytometry in a highly complex institution in the city of Medellín, Colombia during the years 2015 - 2017. Methodology: This is a retrospective descriptive observational study, which included patients with leukemia diagnosed by flow cytometry. A non-probabilistic sampling of consecutive cases was carried out. The information collected was entered into a database in Excel, and the analysis was carried out through the IBM SPSS Version 24 program, using absolute and relative frequencies, average and standard deviation or median and interquartile ranges, according to the nature of each variable and its distribution. Results: 60 patients were included in which male sex predominated with 63.3 percent (38). The most frequent diagnosis was lymphoid leukemia with 78.3 percent (47). Of the total patients included, 36.6 percent (22) were positive for minimal residual disease; 28.3 percent received a bone marrow transplant and 10 percent (6) had a cerebrospinal fluid compromise. In the second cytometry of the patients with minimal residual disease, 90.9 percent (20) expressed CD45 +. 31.8 percent (7) of the patients with minimal residual disease relapsed. Conclusion: Minimal residual disease is a frequent pathology in patients with acute leukemias that requires follow-up and constitutes a relevant prognostic factor(AU)


Subject(s)
Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/prevention & control , Neoplasm, Residual/diagnosis , Flow Cytometry/methods , Epidemiology, Descriptive , Retrospective Studies
9.
Rev. cuba. hematol. inmunol. hemoter ; 37(2): e1297, 2021. graf
Article in Spanish | LILACS, CUMED | ID: biblio-1289428

ABSTRACT

Introducción: La enfermedad granulomatosa crónica es una inmunodeficiencia primaria congénita del sistema inmune innato, originada por defectos en el complejo enzimático nicotinamida adenina dinucleótido fosfato oxidasa presente en células fagocíticas. Estos defectos funcionales causan incapacidad para producir especies reactivas del oxígeno en los fagocitos, que afectan la eliminación de algunos microorganismos patógenos dentro del fagolisosoma. El diagnóstico de esta enfermedad se realiza actualmente mediante la prueba de 1,2,3-dihidrorodamina asistida por citometría de flujo multiparamétrica, o la tinción de fagocitos con nitroazul de tetrazolio asistida por microscopio óptico. Objetivos: Describir los aspectos fisiopatológicos y moleculares de la enfermedad granulomatosa crónica; y discutir aspectos relacionados con las pruebas de diagnóstico antes mencionadas. Métodos: Se realizó una investigación bibliográfica-documental a partir de artículos científicos publicados desde 1933 hasta 2018, para ello fueron consultadas las bases de datos SciELO, PubMed y Springer. Desarrollo: Se exponen las características fisiopatológicas de la enfermedad granulomatosa crónica, así como la relación entre las mutaciones genéticas más abundantes en la población afectada y la gravedad de las manifestaciones clínicas que presentan los pacientes. Además, se analizan críticamente los beneficios y las deficiencias de dos técnicas que se utilizan actualmente para diagnosticar la enfermedad. Conclusiones: La enfermedad granulomatosa crónica puede generar consecuencias inmunológicas e inflamatorias graves, que se hallan en consonancia con las características genéticas expresadas en el complejo enzimático dañado. El diagnóstico de la enfermedad resulta más confiable, exhaustivo y específico, mediante la citometría de flujo y su prueba de 1,2,3-dihidrorodamina(AU)


Introduction: Chronic granulomatous disease is a congenital primary immunodeficiency of the innate immune system, caused by defects in the nicotinamide adenine dinucleotide phosphate oxidase enzyme complex present in phagocytic cells. These functional defects cause inability to produce reactive oxygen species in phagocytes, affecting the elimination of some pathogenic microorganisms within the phagolysosome. The diagnosis of this disease is currently made by means of the 1,2,3-dihydrorodamine test assisted by multiparametric flow cytometry, or the staining of phagocytes with nitro-blue tetrazolium assisted by light microscopy. Objectives: To characterize molecular and pathophysiologically the chronic granulomatous disease; and to discuss aspects related to the aforementioned diagnostic tests. Methods: In this work, a bibliographic-documentary research was carried out from scientific articles published from 1933 to 2018, for which the SciELO, PubMed and Springer databases were consulted. Development: The pathophysiological characteristics of chronic granulomatous disease are exposed, as well as the relationship between the most abundant genetic mutations in the affected population, and the severity of the clinical manifestations presented by the patients. In addition, the benefits and deficiencies of two techniques currently used to diagnose the disease are critically analyzed. Conclusions: Chronic granulomatous disease can generate severe immunological and inflammatory consequences, which are in line with the genetic characteristics expressed in the damaged enzyme complex. The diagnosis of the disease is more reliable, exhaustive and specific, using flow cytometry and its 1,2,3-dihydrorodamine test(AU)


Subject(s)
Humans , Male , Female , Reactive Oxygen Species , Diagnostic Tests, Routine , Nitroblue Tetrazolium/therapeutic use , Diagnostic Techniques and Procedures , Flow Cytometry/methods , Granulomatous Disease, Chronic/physiopathology , Granulomatous Disease, Chronic/genetics
11.
Rev. cuba. hematol. inmunol. hemoter ; 36(4): e1244, oct.-dic. 2020. graf
Article in Spanish | LILACS, CUMED | ID: biblio-1289418

ABSTRACT

Introducción: La citometría de flujo es una técnica de avanzada, objetiva y altamente sensible que permite el análisis y la cuantificación simultánea de múltiples parámetros celulares, muy utilizada en el estudio de la leucemia linfoide crónica, entidad caracterizada como un trastorno proliferativo maligno de linfocitos de aspecto maduro e incompetentes. Objetivo: Describir la estrategia de diagnóstico del inmunofenotipaje por citometría de flujo de la leucemia linfoide crónica. Métodos: Se analizó una muestra de médula ósea para la citometría de flujo de un paciente con sospecha clínica y morfológica de la leucemia linfoide crónica. El inmunofenotipaje celular se realizó con el empleo de anticuerpos monoclonales dirigidos contra los antígenos de diferenciación linfoides B y T. Se procedió a la lectura de la muestra en un citómetro GALLIOS, Beckman Coulter y los datos obtenidos se analizaron con el empleo del programa informático Kaluza. Resultados: Los antígenos con expresión positiva fueron el CD19 (99,94 por ciento), CD20 (81,56 por ciento), CD5 (80,25 por ciento), así como la coexpresión de CD5+/CD19+ (96,56 por ciento), CD5+/CD20+ (80,56 por ciento), CD19+/CD20+ (84,86 por ciento), CD23 (62,65 por ciento), CD49d (65,18 por ciento), CD38 (52,17 por ciento). Se encontró monoclonalidad de la cadena ligera k en un 44,27 por ciento. La expresión de los antígenos CD3, CD4, CD8y CD25 resultó negativa. Conclusiones: La estrategia diagnóstica propuesta permitió identificar los antígenos más frecuentemente expresados en pacientes con leucemia linfoide crónica, así como la coexpresión de los mismos y la monoclonalidad de la cadena k, los cuales son marcadores celulares que permiten realizar el diagnóstico inmunofenotípico de la leucemia linfoide crónica, por citometría de flujo(AU)


Introduction: Flow cytometry is an advanced, objective, highly sensitive technique for the simultaneous analysis and quantification of multiple cellular parameters. This technique is very common in the study of chronic lymphocytic leukemia, a condition defined as a malignant proliferative disorder of mature, incompetent lymphocytes. Objective: Describe the diagnostic strategy for flow cytometry immunophenotyping of chronic lymphocytic leukemia. Methods: Flow cytometry testing was performed of a bone marrow sample taken from a patient with clinical and morphological suspicion of chronic lymphocytic leukemia. Cell immunophenotyping was based on monoclonal antibodies targeted against lymphoid differentiation antigens B and T. The sample was read in a GALLIOS Beckman Coulter cytometer, and the data obtained were analyzed with the software Kaluza. Results: Antigens with a positive expression were CD19 (99.94 percent), CD20 (81.56 percent), CD5 (80.25 percent), as well as the co-expression of CD5+/CD19+ (96.56 percent), CD5+/CD20+ (80.56 percent), CD19+/CD20+ (84.86 percent), CD23 (62.65 percent), CD49d (65.18 percent), CD38 (52.17 percent). Monoclonality of the k light chain was present in 44.27 percent. Expression of antigens CD3, CD4, CD8 and CD25 was found to be negative. Conclusions: The diagnostic strategy proposed made it possible to identify the antigens most frequently expressed in patients with chronic lymphocytic leukemia, as well as their co-expression and the monoclonality of the k chain, all of which are cell markers allowing flow cytometry-based immunophenotypical diagnosis of chronic lymphocytic leukemia(AU)


Subject(s)
Humans , Flow Cytometry/methods , Lymphoma/diagnosis
12.
Rev. cuba. hematol. inmunol. hemoter ; 36(3): e1151, jul.-set. 2020. tab, graf
Article in Spanish | LILACS, CUMED | ID: biblio-1156441

ABSTRACT

Introducción: La citometría de flujo permite la cuantificación de las subpoblaciones de linfocitos con una elevada sensibilidad, especificidad y objetividad. Estas ventajas solo se logran con un proceso laborioso de diseño individualizado y controlado para cada experimento. Objetivo: Diseñar un protocolo de un solo tubo policromático de citometría flujo para inmunofenotipo linfocitario periférico. Métodos: Se realizó un estudio experimental in vitro con muestras de sangre periférica obtenidas de tres voluntarios sanos, en el Centro Nacional de Genética Médica, en marzo de 2019. El tubo se compuso de seis marcadores de linaje para identificar linfocitos B, T, natural killer y natural killer T. Se desarrolló un protocolo de lisis de hematíes sin lavado. Se emplearon anticuerpos monoclonales conjugados con fluorocromos. El punto óptimo de concentración correspondió al mayor índice de tinción y conservación de los porcentajes de positividad de cada población. Se realizó la construcción progresiva del tubo y se propuso una estrategia lógica de secuencia de ventanas para el análisis de datos. Resultados: Los marcadores seleccionados permitieron realizar correctamente el inmunofenotipo linfocitario periférico. En los cinco puntos de titulación se observaron buenas discriminaciones entre las señales positivas y negativas, excepto para el anti-CD56 que presentó una tendencia decreciente del índice de tinción. El volumen total de conjugados requeridos para la determinación de los 6 antígenos fue de 3,75 μL por tubo. Conclusiones: Se obtuvo un tubo policromático que permite el inmunofenotipo periférico de forma rápida y precisa por seis antígenos linfocitarios simultáneamente, con el empleo de pequeños volúmenes de conjugado y sangre(AU)


Introduction: Flow cytometry allows quantification of lymphocyte subpopulations with high sensitivity, specificity and objectivity. These advantages are only achieved through the hardworking process of individualized and controlled design for each experiment. Objective: To design a flow cytometry protocol of a single polychromatic tube for peripheral lymphocyte immunophenotype. Methods: An experimental in vitro study was carried out, in March 2019, with peripheral blood samples obtained from three healthy volunteers, at the National Center for Medical Genetics. The tube was made up of six lineage markers for identifying natural B and T lymphocytes, natural killers and natural killer T cells. A protocol was developed for red blood cell lysis without washing. Fluorochrome-conjugated monoclonal antibodies were used. The optimal point of concentration corresponded to the highest staining index and preservation of the positivity percentages of each population. Progressive tube construction was performed and a logical window sequence strategy was proposed for data analysis. Results: The chosen markers allowed to carry out correct peripheral lymphocyte immunophenotype. Good discriminations between positive and negative signals were observed at the five titration points, except for anti-CD56, which presented a decreasing trend in the staining index. The total volume of conjugates required for determination of the six antigens was 3.75 μL per tube. Conclusions: A polychromatic tube was obtained that allows to carry out peripheral immunophenotype quickly and precisely by six lymphocyte antigens simultaneously, with the use of small volumes of conjugate and blood(AU)


Subject(s)
Humans , Male , Female , Process Optimization , Flow Cytometry/methods , Genetics, Medical , Construction Industry
14.
Rev. cuba. hematol. inmunol. hemoter ; 36(2): e1187, abr.-jun. 2020. tab, graf
Article in Spanish | LILACS, CUMED | ID: biblio-1149900

ABSTRACT

Introducción: La determinación de los inmunofenotipos linfocitarios en sangre periférica forma parte de la evaluación del estado general del sistema inmunitario. Estos exámenes ofrecen informaciones sobre la distribución, concentración y funcionabilidad de las células inmunitarias, lo cual contribuye a establecer pronósticos en el cáncer y predicciones a las respuestas terapéuticas. Objetivo: Evaluar la distribución de las concentraciones linfocitarias circulantes en sangre periférica de pacientes con cáncer. Métodos: Se realizó un estudio analítico en 154 pacientes con cáncer, atendidos en el Instituto de Oncología y Radiobiología de La Habana, durante los años 2017 a 2019. Se empleó la citometría de flujo multiparamétrica para identificar los inmunofenotipos linfocitarios. Este procedimiento se realizó antes de comenzar cualquier tratamiento inmunoterapéutico. Resultados: Los pacientes con cáncer mostraron mayor heterogeneidad en la distribución de las poblaciones linfocitarias respecto a los controles. En los pacientes la mediana de los linfocitos totales y de las subpoblaciones linfocitarias CD3+, CD4+, CD8+ y CD19+ fueron significativamente menores. Los linfocitos T dobles positivos (CD4/CD8) se encontraron elevados significativamente. No se hallaron diferencias entre sexos. La edad se asoció negativamente con las concentraciones de las poblaciones T en tumores sólidos, y con T y B en los linfomas. En el cáncer de próstata se obtuvieron los valores más bajos de poblaciones linfocitarias. Conclusiones: Los pacientes con cáncer tienen menor concentración de linfocitos en sangre periférica que los controles sanos. Las células más afectadas fueron las subpoblaciones T y los linfocitos B. La edad se asoció negativamente con las concentraciones sanguíneas de linfocitos, lo cual pudiera estar en relación con la inmunosenescencia(AU)


Introduction: Determination of lymphocytic immunophenotypes in peripheral blood is part of the evaluation of the general state of the immune system. These tests provide information about the distribution, concentration, and functionality of immune cells, which helps establish prognoses in cancer and predictions of therapeutic responses. Objective: To evaluate the distribution of circulating lymphocyte concentrations in peripheral blood of cancer patients. Methods: An analytical study was carried out with 154 cancer patients treated at the Institute of Oncology and Radiobiology in Havana, from 2017 to 2019. Multiparametric flow cytometry was used to identify lymphocyte immunophenotypes. This procedure was performed before beginning any immunotherapeutic treatment. Results: Cancer patients showed greater heterogeneity in the distribution of lymphocyte populations compared to control patients. The median for total lymphocytes and the lymphocyte subpopulations of CD3+, CD4+, CD8+ and CD19+ were significantly lower in patients. CD4+ CD8+ double-positive T lymphocytes were found to be significantly elevated. No sex differences were found. Age was negatively associated with the concentrations of T-cells populations in solid tumors, and with T- and B-cells populations in lymphomas. In prostate cancer, the lowest values ​​of lymphocyte populations were obtained. Conclusions: Cancer patients have a lower concentration of lymphocytes in peripheral blood than healthy patients in the control group. The most affected ones were the T-cells subpopulations and B lymphocytes. Age was negatively associated with blood levels of lymphocytes, which could be related to immunosenescence(AU)


Subject(s)
Humans , Male , Female , Immunophenotyping/methods , Flow Cytometry/methods , Medical Oncology/methods
15.
Rev. cuba. hematol. inmunol. hemoter ; 36(1): e1137, ene.-mar. 2020. graf
Article in Spanish | LILACS, CUMED | ID: biblio-1126539

ABSTRACT

Introducción: La citometría de flujo es una técnica de avanzada, objetiva y altamente sensible que permite el análisis y la cuantificación simultánea de múltiples parámetros celulares; es muy utilizada en el estudio de las hemopatías malignas. En los últimos años, ha demostrado ser de gran utilidad en la identificación y la caracterización inmunofenotípica de los síndromes linfoproliferativos crónicos. Estos constituyen un grupo heterogéneo de enfermedades caracterizadas por la expansión monoclonal de células linfoides de aspecto maduro. Objetivos: Analizar los aspectos generales de la aplicación de la técnica de citometría de flujo al estudio y clasificación inmunofenotípica de los síndromes linfoproliferativos crónicos. Métodos: Se realizó una investigación bibliográfica-documental acerca del tema. Se consultaron las bases de datos de SciELO y Pubmed. Análisis y síntesis de la información: Se describen los aspectos técnicos de la citometría de flujo, desde la obtención y procesamiento de las muestras hasta la generación del informe por el citómetro; así como la aplicación de la técnica a la caracterización inmunofenotípica de los síndromes linfoproliferativos crónicos. La citometría de flujo multiparamétrica se ha convertido en uno más de los métodos diagnósticos de este síndrome. Uno de los principales objetivos del estudio inmunofenotípico por citometría de flujo consiste en descartar si esa población de células B es clonal o no. Conclusiones: La citometría de flujo permite el análisis, la interpretación y la clasificación inmunofenotípica de los síndromes linfoproliferativos crónicos. Es una herramienta útil en las que se apoya el diagnóstico y el seguimiento de estos pacientes(AU)


Introduction: Flow cytometry is an advanced, objective and highly sensitive technique that allows simultaneous quantification and analysis of multiple cellular parameters. It is widely used in the study of malignant hemopathies. In recent years, it has proved very useful in the identification and immunophenotypic characterization of chronic lymphoproliferative syndromes. These conditions belong to a heterogeneous group of diseases characterized by monoclonal expansion of mature lymphoid cells. Objectives: To analyze the general aspects of flow cytometry application to the study and immunophenotypic classification of chronic lymphoproliferative syndromes. Methods: A bibliographic-documentary research about the topic was carried out. We consulted the SciELO and Pubmed databases. Information analysis and synthesis: The technical aspects of the flow cytometry are described, from obtaining and processing the samples to the cytometer's generating the report; as well as the technique's application to the immunophenotypic characterization of chronic lymphoproliferative syndromes. Multiparametric flow cytometry has become one of the diagnostic methods for this syndrome. One of the main objectives of the immunophenotypic study by flow cytometry is to rule out whether this population of B cells is clonal or not. Conclusions: Flow cytometry allows the analysis, interpretation and immunophenotypic classification of chronic lymphoproliferative syndromes. It is a useful tool that supports the diagnosis and monitoring of these patients(AU)


Subject(s)
Humans , Immunophenotyping/methods , Flow Cytometry/methods , Lymphoproliferative Disorders/diagnostic imaging
16.
Einstein (Säo Paulo) ; 18: eAO5236, 2020. graf
Article in English | LILACS | ID: biblio-1133772

ABSTRACT

ABSTRACT Objective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.


RESUMO Objetivo Acompanhar a expansão de células-tronco mesenquimais de cordão umbilical por dois marcadores clássicos de senescência, p16 (INK4A) e p21 (CDKN1A), usando métodos práticos, rápidos e com custo menor do que a técnica padrão-ouro de Western blotting, para avaliar sua aplicabilidade em laboratório. Métodos Células-tronco mesenquimais de cordão umbilical foram isoladas da geleia de Wharton e, após controle de qualidade e caracterização morfológica e imunofenotípica por citometria de fluxo, foram expandidas em cultura, até chegarem próximas à parada do ciclo celular (senescência replicativa). Resultados Foi feita a comparação entre células jovens, na passagem 5, e células pré-senescentes, na passagem 10, avaliando a expressão proteica dos marcadores clássicos de senescência celular p16 e p21, comparando os resultados obtidos por Western blotting com os obtidos por citometria de fluxo e imunofluorescência indireta. Conclusão O seguimento de culturas celulares, por meio da imunofluorescência indireta de p16, permite identificar as culturas de células-tronco mesenquimais de cordão umbilical em risco de atingirem a senescência replicativa.


Subject(s)
Humans , Umbilical Cord/physiology , Fluorescent Antibody Technique/methods , Cellular Senescence , Mesenchymal Stem Cells/physiology , Flow Cytometry/methods , Biomarkers/blood , Cells, Cultured , Blotting, Western , Cyclin-Dependent Kinase Inhibitor p16 , Cyclin-Dependent Kinase Inhibitor p21
17.
Einstein (Säo Paulo) ; 18: eAO4560, 2020. graf
Article in English | LILACS | ID: biblio-1101099

ABSTRACT

ABSTRACT Objective To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. Methods Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. Results Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. Conclusion The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.


RESUMO Objetivo Avaliar o efeito dos compostos ICI 182,780 (fulvestranto), um antagonista seletivo dos receptores de estrógeno alfa/beta (REα/REβ), e do G-1, um agonista seletivo de receptores de estrógeno acoplados a proteínas-G (GPER), na possível indução de autofagia em linhagens de câncer de mama MCF-7 e SKBr3, bem como o efeito de G-1 na viabilidade celular. Métodos A viabilidade celular de células MCF-7 e SKBr3 foi avaliada pelo ensaio com MTT. Para investigar a indução da autofagia, células MCF-7 foram transfectadas com GFP-LC3, um marcador de autofagossomos, e analisadas por microscopia de fluorescência em tempo real. As células MCF-7 e SKBr3 foram incubadas com o indicador de compartimentos ácidos laranja de acridina e analisadas por citometria de fluxo como indicativo para autofagia. Resultados Em células MCF-7, o ICI 182,780 e rapamicina após 48 horas levaram à diminuição da viabilidade celular, enquanto o G-1 não alterou a viabilidade no mesmo período de tratamento. Nem o ICI 182,780 e nem o G-1 induziram aumento na pontuação de GFP-LC3 em células MCF-7 até 4 horas. Já os ensaios de citometria de fluxo demonstraram que ICI 182,780 levou ao aumento de compartimentos ácidos em células MCF-7. O G-1 não aumentou estes parâmetros em ambas as linhagens. Por outro lado, ICI 182,780 e G-1 não induziram à redução da viabilidade em células SKBr3 e nem à formação de compartimentos ácidos, como etapa final do processo autofágico. Conclusão O aumento de compartimentos ácidos pelo ICI 182,780 em células de câncer de mama positivas para receptores de estrógeno parece estar associado com seu efeito inibidor de receptores de estrógeno, mas sem o envolvimento de GPER. A compreensão desses mecanismos pode direcionar estudos sobre o envolvimento dos receptores nos processos celulares de resistência do câncer de mama.


Subject(s)
Humans , Female , Autophagy/drug effects , Breast Neoplasms/pathology , Breast Neoplasms/drug therapy , Receptors, G-Protein-Coupled/agonists , Estrogen Receptor Antagonists/pharmacology , Fulvestrant/pharmacology , Time Factors , Transfection/methods , Cell Survival/drug effects , Blotting, Western , Reproducibility of Results , Analysis of Variance , Sirolimus/pharmacology , Receptors, G-Protein-Coupled/analysis , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor beta/antagonists & inhibitors , Cell Proliferation/drug effects , MCF-7 Cells , Flow Cytometry/methods
18.
Einstein (Säo Paulo) ; 18: eAO4966, 2020. tab, graf
Article in English | LILACS | ID: biblio-1056043

ABSTRACT

ABSTRACT Objective To validate multilineage score system correlating results of flow cytometry, cytogenetics, cytomorphology and histology from samples of patients with suspected myelodysplastic syndrome or cytopenia of unknown origin. Methods A retrospective study analyzing laboratory data of 49 patients with suspected myelodysplastic syndrome or cytopenia of unknown origin, carried out between May and September 2017. The inclusion criteria were availability of flow cytometry results, and at least one more method, such as morphology, histology or cytogenetics. Thirty-eight patients were classified as diagnosis of myelodysplastic syndromes, whereas 11 were classified as normal. Patients were evaluated based on score systems, Ogata score and flow cytometry multilineage score. Results Comparing the scores obtained in the Ogata score and the multilineage score, it was observed that in four cases the Ogata score was zero or 1 point, while the multilineage score was higher than 3 points. In addition, in 12 cases with Ogata score of 2, the multilineage score was greater than 3. Conclusion The flow cytometry multilineage score system demonstrated to be more effective in dysplasia analysis, by assessing the erythroid, monocytic, granulocytic and precursor cell lineages, apart from the parameters evaluated by the Ogata score.


RESUMO Objetivo Validar ficha de escore multilinhagem correlacionando resultados obtidos de citometria de fluxo, citogenética, citomorfologia e histologia de amostras de pacientes com suspeita de síndrome mielodisplásica ou citopenias a esclarecer. Métodos Estudo retrospectivo de análise de dados laboratoriais de 49 pacientes com suspeita clínica de síndrome mielodisplásica ou citopenias a esclarecer realizado entre maio e setembro de 2017. Os critérios de inclusão foram a disponibilidade de resultados de citometria de fluxo e de, pelo menos, outra metodologia, entre morfologia, histologia, ou citogenética. Trinta e oito pacientes foram classificados como diagnosticados com síndromes mielodisplásicas enquanto 11 foram classificados como normais. Os pacientes foram avaliados utilizando sistemas de escore, escore de Ogata e ficha multilinhagem. Resultados Comparando as pontuações obtidas no escore de Ogata e na ficha multilinhagem, observou-se que, em quatro casos, o score de Ogata foi zero ou 1 ponto, enquanto, pela ficha multilinhagem, a pontuação foi superior a 3 pontos. Além disso, em 12 casos com escore de Ogata 2, a pontuação pela ficha multilinhagem foi superior a 3. Conclusão A ficha multilinhagem demonstrou ser mais eficaz na análise de displasia por avaliar as linhagens eritroide, monocítica, granulocítica e células precursoras, além dos parâmetros avaliados no escore de Ogata.


Subject(s)
Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Adult , Aged , Aged, 80 and over , Young Adult , Myelodysplastic Syndromes/pathology , Flow Cytometry/standards , Reference Standards , Biopsy , Bone Marrow Cells/pathology , Monocytes/pathology , Reproducibility of Results , Retrospective Studies , Cytogenetic Analysis/methods , Cytogenetic Analysis/standards , Erythroid Cells/pathology , Flow Cytometry/methods , Granulocytes/pathology , Middle Aged
19.
Braz. oral res. (Online) ; 34: e033, 2020. graf
Article in English | LILACS | ID: biblio-1089391

ABSTRACT

Abstract The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.


Subject(s)
Humans , Phenotype , Stem Cells/cytology , Keratinocytes/cytology , Cell Culture Techniques/methods , Epithelial Cells/cytology , Mouth Mucosa/cytology , Receptors, Transferrin/analysis , Biomarkers/analysis , Antigens, CD/analysis , Cell Separation/methods , Reproducibility of Results , Receptors, Nerve Growth Factor/analysis , Flow Cytometry/methods , Nerve Tissue Proteins/analysis
20.
Rev. cuba. hematol. inmunol. hemoter ; 35(4): e1123, oct.-dic. 2019. tab, graf
Article in Spanish | LILACS, CUMED | ID: biblio-1093297

ABSTRACT

Introducción: El cáncer epitelial de ovario (CEO) ocupa el sexto lugar en incidencia y mortalidad a nivel mundial y en Cuba, el quinto en incidencia. Este cáncer es inmunogénicoy sus células malignas crecen en interacción conlas células inmunitarias. Su curso clínico depende del infiltrado inflamatorio acompañante del tumor. La citología e histopatología son los métodos diagnóstico de elección. Sin embargo, la citometría de flujo emerge como una tecnología de mayor sensibilidad, objetividad y rapidez. Objetivo: Diseñar un panel multicolor de citometría de flujo para inmunofenotipar el infiltrado linfocitario de tres tipos de muestras de pacientes con CEO. Métodos: Se realizó un diseño experimental, para la creación y evaluación de un panel multicolor de citometríade flujo, en el laboratorio de Inmunología del Instituto Nacional de Oncología y Radiobiología. El panel se diseñó en sangre de 3 sujetos sanos y se optimizó para sangre periférica en 33 sujetos sanos y, en sangre periférica, ascitis y tejido tumoral ovárico de tres pacientes con CEO. En cada muestra se inmunofenotiparon varias poblaciones linfocitarias. Resultados: Se seleccionaron 11 marcadores antigénicos para el inmunofenotipo, el panel quedó conformado por 4 tubos de citometría. La metodología se pudo aplicar a las muestras de ascitis y tejido tumoral sin interferencias, se obtuvieron porcentajes de las subpoblaciones linfocitarias dentro de los valores esperados. Conclusiones: El panel diseñado permitió inmunofenotipar linfocitos en distintos tipos de muestras de pacientes con CEO, con resultados confiables y reproducibles. Esta metodología puede extenderse a la realización de inmunofenotipaje en otras enfermedades(AU)


Introduction: Epithelial ovarian cancer occupies the 6th place in incidence and mortality in women worldwide. In Cuba, it occupies the 5th place in incidence in females. This cancer is immunogenic and its malignant cells grow in interaction with multiple cells from immune system. Its clinical course depends largely on the type of inflammatory infiltrate accompanying the tumor. Cytology and histopathology are gold standard as diagnostic methods. However, flow cytometry emerges as a technology with greater sensitivity, objectivity and speed. Objective: To design a multicolored flow cytometry panel to immunophenotype the lymphocytic infiltrate of three types of samples for patients with ovarian cancer. Methods: An experimental design was carried out in vitro for the creation and evaluation of a multicolored flow cytometry panel in the Immunology laboratory of the National Institute of Oncology and Radiobiology of Cuba. The panel was designed in the blood of three healthy subjects; then it was optimized for blood in 33 healthy volunteers and blood, ascites and ovarian tumor tissue, from three patients with epithelial ovarian cancer. Several lymphocytes lineages were immunophenotypedin each sample. Results: Eleven markers were selected for the immunophenotype and the panel was made up of four multiparameter cytometry tubes. The methodology created could be applied to the samples of ascites and tumor tissue without interferences and percentages of different lymphocyte subpopulations were obtained within the expected values. Conclusions: The designed panel allowed immunophenotyping of lymphocytes in different types of ovarian cancer patient samples and reliable and reproducible results were obtained. This methodology could be employed for others diseases(AU)


Subject(s)
Humans , Female , Flow Cytometry/methods , Immunophenotyping/methods , Equipment Design/methods , Carcinoma, Ovarian Epithelial/diagnosis
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