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- ItemAcesso aberto (Open Access)Associação entre os alelos HLA-DRB1 e a aloimunização eritrocitária(Universidade Federal de São Paulo (UNIFESP), 2019-02-13) Souza, Conceicao Pinheiro De [UNIFESP]; Bordin, Jose Orlando [UNIFESP]; http://lattes.cnpq.br/4235368036147314; http://lattes.cnpq.br/4350403289378059; Universidade Federal de São Paulo (UNIFESP)Background: The immunogenicity of antigens from blood group systems is now better understood by the availability of knowledge of the genetic basis of the proteins involved, molecular orientation in the erythrocyte membrane and the nature of cellular immune responses. Aims: To investigate the association of the HLA-DRB1 alleles in the selective immune response against the antigens of the RH, KEL, FY, JK and MNS systems, either in the sensitization against one or multiple erythrocyte antigens, as well as to verify if there is any allele associated with a protective effect alloimmunization. Methods: This study that analyzed blood samples from 215 patients alloimmunized against antigens from the RH, KEL, FY, JK and MNS systems and compared them with three control groups: 100 blood donors, 101 non-alloimmunized patients and 174,015 candidates for bone marrow donation. Alloantibodies were identified by gel methodology and confirmed by erythrocyte phenotyping. The HLA-DRB1 genotyping was performed by the PCR- SSO technique, using Luminex technology. The GraphPad InStat software was used for statistical analysis. The in silico analysis was performed using the NetMHCIIpan-3.2 server, able to predict the possibilities of interaction between the amino acid sequences of the RhD, RhCE, KEL, FY, JK and MNS proteins and the polymorphic pockets residues of the DRB1 molecules. Alignment was used to evaluate the similarity between the residues located along the β1 domain of the HLA-DR molecules, which showed strong interaction with the anchor peptides derived from the antigenic proteins. Results: Among the multialoimmunized patients, the anti-D+C association was the most frequent, present in 90.3% of the associations with anti-D. Statistical analysis indicated the association between the HLA-DRB1*01 allele and the anti-C and anti-Jka alloimmunizations, as well as the HLA-DRB1*15 allele in the comparison between patients with single and multiple clinically relevant red blood cell antibodies and bone marrow donors control group. This latter finding was due to the association of the HLA-DRB1*15 allele with the anti-D, -C, -E and -K aloimmunizations. The in silico analysis confirmed the associations shown, demonstrating a strong interaction between the DRB1*01 molecule and the anchor peptides derived from the RhCe and JK proteins, and between the DRB1*15 molecule and the major anchor peptides derived from RhD, RhCe, RhcE and KEL proteins, which contained the polymorphic determinants of D, C, E and K antigens in their sequences or adjacent regions. Although the HLA-DRB1*11 allele had a higher frequency among bone marrow donors, apparently exerting a protective effect on alloimmunization, in silico analysis refuted this finding. While the protein sequence containing the 193M polymorphism of the K antigen showed high histocompatibility promiscuity with numerous DRB1 molecules, the sequence containing the 280D polymorphism of the Jka antigen showed interaction only with the DRB1*01 molecule, and the sequences containing the polymorphisms of Fya (42G ) and S (29M) showed weak binding with few molecules. Conclusion: The anti-D+C association, whose frequency is high among alloimmunized patients, could be explained by two factors: the high phenotypic frequency of D and C antigens in the population and the interaction capacity of several DRB1 molecules, such as DRB1*12:01, *15:01 and *15:03, with the anchor peptides derived from the RhD and RhCe proteins, which contained 60I, 68S and 103S polymorphisms common to D and C antigens. The difference in reactivity between the DRB1 molecules and the JK, FY and MNS proteins observed in the in silico analysis seems to be directly associated with the low immunogenicity of the Jka, Fya and S antigens, compared to the D, C, E and K antigens. In silico analysis of blood group proteins may be a practical resource, at no additional cost to the laboratory, which allows a greater understanding of the interaction capacity between antigenic peptides and HLA-DR molecules in the presentation process and consequent production of alloantibodies.
- ItemAcesso aberto (Open Access)Bomba de infusão por seringa para transfusão de concentrados de hemácias: análise dos níveis de marcadores de hemólise(Universidade Federal de São Paulo (UNIFESP), 2016-12-21) Gannam, Fernanda Figueiredo [UNIFESP]; Pedreira, Mavilde da Luz Goncalves [UNIFESP]; http://lattes.cnpq.br/5901248667753975; http://lattes.cnpq.br/7966962702628127; Universidade Federal de São Paulo (UNIFESP)Introduction: Syringe infusion pumps (SIP) were developed in the second half of the twentieth century, leading to major advances in intravenous therapy, and they were gradually applied to the transfusion of packed red blood cells (RBC), raising questions on possible cells damage. Objectives: Determining levels of hematocrit levels, total hemoglobin, free hemoglobin, lactate dehydrogenase (LDH), potassium and hemolysis percentage of RBC administered by SIP; investigating the influence of the infusion rate on hemolysis markers levels and describing the influence of RBC storage time, ambient temperature and humidity on hemolysis markers levels. Methods: Experimental study, conducted with 14 packed RBC, of which blood aliquots were filled into syringes. Three SIP of a same manufacture were randomly studied and infusion rates of 5 ml/h, 10 ml/h and 20 ml/h. The hemolysis markers analyzed were: hematocrit (%), total hemoglobin (g/dl), free hemoglobin (g/dl) LDH (U/l), potassium (mmol/l) and hemolysis degree (%). The variables were analyzed according to the average, standard deviation and median, applying the ANOVA or Friedman tests, drawning a linear correlation and coefficients according to Pearson or Spearman?s methods (p=0.05). Results: It was performed 324 hemolysis markers analyses. Among all the analyses that were performed on RBC before manipulation, after filling the syringe and after infusion by SIP, a decrease of total hemoglobin level (p=0.003) was identified with an increase of free hemoglobin level (p<0.001) and on the hemolysis percentage (p<0.001). The comparisons of infusion rates have shown an increase of free hemoglobin levels (5 ml/h: p=0.030; 10 ml/h: p=0.002; 20 ml/h: p=0.016) and hemolysis percentage (5 ml/h: p=0.006; 10 ml/h: p=0.002; 20 ml/h: p=0.009) in all the evaluated rates and there was a significant reduction of total hemoglobin at 10 ml/h (p=0.001). The variations in potassium concentration (p=0.030; p=0.030) and hematocrit (p=0.029; p=0.001) were correlated, respectively, to the ambient temperature and humidity, and temperature with LDH (p=0.008) variation. The RBC storage time was moderately correlated with changes on hemolysis degree (p<0.001), potassium levels (p=0.005) and free hemoglobin (p=0.009). Conclusion: RBC infused by SIP presented changes in free hemoglobin and hemolysis degree levels in all studied infusion rates, and a decrease of total hemoglobin at 10 ml/h. The blood aliquoting into syringes has created a significant increase in free hemoglobin level and the hemolysis degree, indicating occurrence of cell lysis during syringes filling. The increase in potassium concentration was directly proportional to the RBC storage time. In addition, it was identified a correlation between an increase in ambient temperature with an increase in potassium levels, hematocrit and LDH.
- ItemEmbargoEfeito do tratamento materno com dexametasona em fígado fetal de ratos: o papel de macrófagos no metabolismo do ferro e maturação de eritroblastos(Universidade Federal de São Paulo (UNIFESP), 2010-11-24) Neves, Flavia Macedo de Oliveira [UNIFESP]; Miraglia, Sandra Maria [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Macrophages (MƒÖs) have important roles during development as a phagocytosis of apoptotic cells, secretion of growth factors and cytokines as well as angiogenesis induction. In the fetal liver (FL), erythroblasts at various stages of differentiation associate with central MƒÖs to form erythroblastic islands (EI). During erythropoiesis, central MƒÖs regulate the proliferation and differentiation of erythroblasts by supplying of growth factors and nutrients as iron to heme synthesis. In addition, central MƒÖs have been shown to produce erythropoietin, the principal factor in the regulation of erythropoiesis. In addition, these cells recognize and uptake extruded erythroblast nuclei. Macrophage differentiation and erythropoiesis can be affected by glucocorticoids (GCs). During the pregnancy, GCs are frequently used to accelerate fetal lung maturation and other organs in the preterm delivery. Thus, in these work we examined changes in the central MƒÖs and erythroblasts of EI after maternal treatment with synthetic GCs. To investigate this, female Wistar rats were mated and treated between 13 to 16 dpc with dexamethasone 21-phosphate (100 ƒÝg/Kg BW/day) or vehicle (control - 0.9% NaCl). At 17 dpc, pregnant rats were euthanized and fetuses were collected to liver isolation. Fetal livers (FL) were processed to light and transmission electron microscopy. Sections (2 £gm) of FL embedded in glycol methacrylate were stained with PAS to determine the erythroid cell (proerythroblasts, basophilic, polychromatophilic and orthochromatic erythroblasts) frequencies. Total cell number from each EI was significant reduced in treated animals. To iron detection, tissue was embedded in paraffin, cut at 5 £gm and stained by Perls+DAB method. Iron accumulation was more evident in MƒÖs from FL of treated fetuses. To confirm this, areas staining positive for iron were converted to pixels and analyzed by software. Ultrastructural observations also showed electron dense granular material similar at iron deposits in cells from IE from treated fetuses. The same was not observed in the control. Iron deposits in central MƒÖs and erythroblasts were confirmed by staining sections with bismuth subnitrate. To test if GCs may induce changes in the cellular iron storage we used rat peritoneal macrophages treated with dexamethasone (10 nM) for 24 h. These cells were incubated with FeCl3 (100 £gM) for 4 h and stained by Prussian blue method. Dexamethasone treated-MƒÖs exhibited greatest amount of intracellular iron than untreated cells confirmed by blue granules quantification. Taken together, these results suggest that dexamethasone can interfere in iron uptake and/or release by macrophages and in the proliferation of erythroblasts affecting the fetal erythropoiesis in rats.
- ItemAcesso aberto (Open Access)Infusão de concentrado de hemácias por cateter central de inserção periférica com válvula proximal: influência sobre os marcadores de hemólise(Universidade Federal de São Paulo (UNIFESP), 2016) Jacinto, Amanda Karina de Lima [UNIFESP]; Avelar, Ariane Ferreira Machado [UNIFESP]; Pedreira, Mavilde da Luz Gonçalves [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introdução: A terapia transfusional pode ser implementada por meio de diversos cateteres, dentre eles o cateter central de inserção periférica (CCIP). Entretanto, evidencia-se carência de estudos que indiquem sua influência sobre alterações nos marcadores de hemólise. Objetivos: Verificar a ocorrência de alterações nos marcadores de hemólise de concentrados de hemácias após infusão por CCIP com válvula proximal, segundo calibre e velocidade de infusão, e descrever a altura do sistema, a pressão e a velocidade de infusão segundo os diferentes calibres de cateter. Material e Método: estudo experimental realizado em laboratório sob condições controladas de temperatura e umidade. A amostra foi composta por 12 CCIP com válvula proximal de 3Fr e 4Fr, nos quais foram infundidos concentrados de hemácias, pelo método gravitacional, e coletadas alíquotas de sangue da bolsa, após a infusão em fluxo livre e em velocidade controlada. As variáveis de desfecho foram porcentagem do hematócrito, hemoglobina total, hemoglobina livre, grau de hemólise, potássio e desidrogenase lática. As variáveis categóricas foram descritas em valor absoluto e frequência relativa enquanto as variáveis contínuas como média, desvio-padrão, mediana, valores mínimo e máximo. Foram utilizados os testes ANOVA, Bonferroni, Mann-Whitney e correlação de Spearman, com nível de significância p<0,05. Resultados: após as infusões nos CCIP de calibre 3 Fr evidenciou-se aumento do grau de hemólise (p=0,003) e da hemoglobina livre (p=0,014), e redução da hemoglobina total (p=0,002), com influência significante das velocidades mínima e máxima sobre grau de hemólise, hemoglobina total na velocidade mínima e hemoglobina livre na velocidade máxima. Quanto à altura do sistema de infusão, pressão e velocidade de infusão, segundo o calibre do CCIP, identificou-se que apenas a velocidade diferiu significativamente entre os dois calibres após infusão em fluxo livre. Conclusões: o menor calibre do cateter e a velocidade de infusão influenciaram variações de alguns marcadores de hemólise, e apenas a velocidade diferiu entre os diferentes calibres de cateter. As alterações evidenciadas nos marcadores de hemólise não contraindicariam a infusão de concentrados de hemácias por CCIP com válvula proximal de calibres 3 e 4 Fr, e em diferentes velocidades de infusão
- ItemSomente MetadadadosInfusion pumps and red blood cell damage in transfusion therapy: an integrative revision of the academic literature(Wiley, 2016) Wilson, Ana Maria Miranda Martins; Peterlini, Maria Angélica Sorgini [UNIFESP]; Pedreira, Mavilde da Luz Gonçalves [UNIFESP]Objectives: to obtain information from scientific literature concerning infusion pumps used in administering erythrocyte (red blood cells) and to evaluate the implications in the practical use of this equipment by nurses when conducting transfusions. Method: an integrative revision of the following scientific databases: Pubmed/Medline, Scopus, the Virtual Library for Health, SciELO, Web of Science and Cochrane. The following descriptors were used: "infusion pumps", "blood transfusion", "transfused erythrocyte" and "hemolyis". There were no restrictions on the scope of the initial data and it was finalized in December 2014. 17 articles were identified in accordance with the inclusion and exclusion criteria. Results: all of the publications included in the studies were experimental in vitro and covered the use of infusion pumps in transfusion therapy. A summary of the data was presented in a synoptic chart and an analysis of it generated the following categories: cellular damage and the infusion mechanism. Conclusion: infusion pumps can be harmful to erythrocytes based on the infusion mechanism that is used, as the linear peristaltic pump is more likely to cause hemolysis. Cellular damage is related to the plasmatic liberation of markers that largely dominate free hemoglobin and potassium. We reiterate the need for further research and technological investments to guide the development of protocols that promote safe practices and that can contribute to future clinical studies.
- ItemSomente MetadadadosMarcadores de hemólise de concentrados de hemácias administrados in vitro por cateter central de inserção periférica: influência da velocidade de infusão, tempo de armazenamento e calibre do cateter(Universidade Federal de São Paulo (UNIFESP), 2019-09-26) Souza, Adja Havreluk Paiva De [UNIFESP]; Avelar, Ariane Ferreira Machado [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introduction: The use of the central peripheral insertion catheter (CCIP) is expanding and can be used even for the administration of blood components. In hospital practice, the use of CCIP is common, but the use of these devices for transfusion therapy is not yet fully supported in the literature, due to the risk of hemolysis due to the mechanical action generated on erythrocytes. Objectives: To identify changes in the levels of hemolysis markers after infusion of red blood cells (RBC) in peristaltic infusion pump by central catheter of peripheral valve insertion, according to time of storage of the blood component bag, infusion rate, and catheter caliber. Method: Experimental study carried out in laboratory under controlled temperature and humidity conditions. The data collection took place after approval of the ethical merit of the research (opinion nº 3.231.723). Concentrated positive A-type RBC concentrates were collected from CPDA-1 provided by the nonprofit Charity Blood Collection Association (COLSAN). RBC concentrates with storage time greater than and less than 14 days were randomly administered in 6 units of 5French (FR) CCIP and 6 4FR units of carbotane, with a PASV (Pressure Activated Safety Valve) valve, 60 cm in length, at speeds 50 and 100ml / h per linear peristaltic pump. Aliquots were analyzed in three moments: A1: - Aliquot of the RBC bag (directly from the packed red blood cells), A2- Aliquot after filling of the system by free flow of the infusion system and A3- Aliquot after controlled infusion. The hemolytic markers analyzed were hemolysis (%), hematocrit - Ht (%), total hemoglobin - Hbt (g / dl), free hemoglobin - Hbl (g / dl), potassium - K lactate dehydrogenase - LDH (U / L). Data were analyzed according to descriptive and inferential statistics, using the T-student, Kruskal-Wallis or Mann-Whitney tests (p <0.05). Results: 48 experiments were performed, and 144 aliquots of red blood cell concentrates were analyzed from the three moments studied. In relation to the infusion rates studied, there was a trend towards an increase in the median values (minimum and maximum) at a rate of 50 ml / h between Hbt A1-A3 moments [of 23,18 (15,70 / 32,49 ) g / dl for 23.99 (16.45 / 30.75 g / dl), Hbl [from 0.11 (0.04 / 0.23) g / dl to 0.12 (0.00 / , 24) g / dl], Ht [from 74.00 (68.00 / 80.00)% to 75.00 (70.00 / 84.00)%], K [ / 39.95) mmol / l for 26.45 (0.10 / 41.63) mmol / l] and LDH [from 1036.16 (0.00 / 2997.85) U / L to 1083.38 (137 , 61 / 5847.29) U / L] and only Ht [from 74.00 (68.00 / 80.00) g / dl to 75.00 (68.00 / 80.00) g / dl] LDH 787.91 [((5.40 / 4600.65) U / L for 835.13 (0.00 / 2879.12) U / L] in 100 ml / hr; and reduction in markers of hemolysis [from 0.13 (0.04 / 0.44)% to 0.12 (0.00 / 0.37)%] at 50ml / h and at 100ml / h Hbt [from 23, 26 (17.10 / 32.50) g / dl to 22.96 (16.48 / 31.53) g / dl] and K xvi [24.95 (0.10 / 41.91) mmol / l for 24.22 (0.05 / 41.16) mmol / l]. With regard to the storage time, there was an upward trend in Hbt, Ht and LDH values in pockets of less than 14 days, and of Ht, K and LDH in pockets with more than 14 days of storage. The tendency to decrease was observed in the markers: Hbl, degree of hemolysis and K in pockets stored for less than 14 days; and in Hbt, Hbl and degree of hemolysis in the pockets with more than 14 days. As for the caliber of the catheters, the same tendency of increase in the values of the Hbt, Ht and LDH markers was observed in both calibers, and opposite tendencies in the K, in which in 4FR there was increase and in 5FR reduction. When comparing the A3 moments, a significant difference was identified between the storage times of the pockets in all the markers, with the exception of Hbt; between gauges, except Hbl; and only in the comparison of infusion rates did not identify significant influence on the markers. Conclusion: The storage time of the pockets greater than 14 days and the caliber 4 Fr of the CCIP valve significantly influenced the levels of Hb t, potassium and LDH. No influence of the infusion rates studied on hemolysis markers was identified. The levels of hemolysis markers identified after the experimental conditions imposed do not contraindicate the administration of CH, according to the recommendations of the competent organs.
- ItemAcesso aberto (Open Access)Marcadores de hemólise em concentrados de hemácias administrados por bombas de infusão peristálticas lineares(Universidade Federal de São Paulo (UNIFESP), 2015-09-30) Martins, Ana Maria Miranda [UNIFESP]; Pedreira, Mavilde da Luz Goncalves Pedreira [UNIFESP]; Peterlini, Maria Angélica Sorgini [UNIFESP]; http://lattes.cnpq.br/1599622257763420; http://lattes.cnpq.br/5901248667753975; http://lattes.cnpq.br/2077514190370554; Universidade Federal de São Paulo (UNIFESP)Introdução: A administração de hemocomponentes por bombas de infusão não é prática totalmente respaldada na literatura, devido ao risco de hemólise pela ação mecânica gerada nos eritrócitos. Objetivos: Determinar e analisar o nível de marcadores de hemólise em concentrados de hemácias administrados por bombas de infusão peristálticas lineares em duas velocidades de infusão. Método: Estudo experimental, com coleta dos dados realizada em laboratório sob condições controladas e após aprovação do Comitê de Ética em Pesquisa. Para o cálculo da amostra, foi feita a estimativa de volume de sangue necessário para o preenchimento dos equipos, dos extensores, para simulação da prática transfusional nas velocidades estudadas e para as análises laboratoriais de cada etapa do experimento. Totalizaram-se quatro unidades de concentrados de hemácias excedentes de estoque fornecidas por um hemocentro. Foram selecionadas bombas de infusão de dois fabricantes, denominadas como A e B, analisadas em triplicata, totalizando seis equipamentos. Para seleção das velocidades de infusão foram mimetizadas situações da prática clínica de enfermagem na terapia transfusional, definidas em 100mL/h e 300mL/h. Houve randomização dos equipamentos, de acordo com as velocidades de infusão. Coletaram-se amostras de controle e experimentais para análise dos marcadores: potássio, hematócrito, hemoglobina total, hemoglobina livre e grau de hemólise. Para análise dos dados, as variáveis contínuas foram descritas por meio da média, desvio-padrão, valores mínimo e máximo e mediana. A distribuição dos marcadores foi estudada por meio da análise de variância (ANOVA) para medidas repetidas e empregaram-se testes de comparações múltiplas entre os momentos do estudo. Foram considerados achados estatisticamente significativos aqueles com probabilidade de erro tipo I inferior ou igual a 5%. Resultados: Das 180 análises dos marcadores estudados, o potássio apresentou alterações significativas na avaliação geral das bombas de infusão e velocidades estudadas (p<0,01). Quando analisadas as velocidades de infusão, o potássio aumentou nos experimentos a 300mL/h (p=0,021). A hemoglobina livre apresentou tendência de elevação em fluxo de 100mL/h (p=0,068). Na análise dos equipamentos, a hemoglobina livre apresentou aumento no experimento com a bomba de infusão A (p=0,026) e o potássio com a B (p=0,022). Os marcadores hematócrito, hemoglobina total e grau de hemólise não apresentaram incrementos estatisticamente significantes nos experimentos. Entretanto, o grau de hemólise apresentou valor acima do recomendado pela legislação vigente entre as etapas do experimento com o fluxo de 300mL/h. Conclusões: Identificou-se potencial para hemólise durante os experimentos, com incremento significante dos marcadores hemoglobina livre e potássio. O potássio aumentou, predominantemente na bomba de infusão B e no fluxo de 300mL/h. O nível de hemoglobina livre mostrou-se elevado nos experimentos com a bomba de infusão A, com tendência de aumento no fluxo de 100mL/h. Considera-se que a realização de novos estudos com outros marcadores de lesão celular e com maior número de amostras sejam relevantes para fundamentação da prática clínica de enfermagem na administração de concentrados de hemácias.
- ItemSomente MetadadadosMulti-target detection of oxidative stress biomarkers in quercetin and myricetin treated human red blood cells(Royal soc chemistry, 2016) Maurya, Pawan Kumar [UNIFESP]; Kumar, Prabhanshu; Nagotu, Shirisha; Chand, Subhash; Chandra, PranjalQuercetin and myricetin are important dietary flavonoids with potential health benefits and interfere with reactive oxygen species metabolism. The objective of this study was multi-target spectroscopic analysis of oxidative stress biomarkers (malondialdehyde (MDA), glutathione (GSH) and sulfhydryl (-SH) groups) in quercetin and myricetin treated red blood cells (RBCs) during human aging. The study was carried out on clinically relevant blood samples obtained from 105 healthy subjects between the ages of 18-82 years. The subjects were divided into three age groups, young (18-35 years), middle (36-60 years) and old (>60 years). Oxidative stress was induced in vitro by incubating RBCs with 10(-5) M tert-butyl hydroperoxide (t-BHP). The effects of flavonoids were evaluated by detecting MDA, GSH and -SH groups by co-incubating the RBCs in the presence of flavonoid (10(-8) M to 10(-5) M final concentration) and t-BHP. The GSH/GSSG ratios were estimated to demonstrate the antioxidant power of the RBCs. The results showed elevated MDA levels (p < 0.001) after incubation with t-BHP as compared to a control. The GSH and -SH groups significantly (p < 0.001) decreased when incubated with t-BHP. In vitro administration of both flavonoids significantly attenuated the deleterious effect of oxidative stress in erythrocytes from all age groups. We showed a significant (p < 0.0001) negative correlation (r = -0.8334) between GSH/GSSG during human aging. We believe that these findings are novel and will help in the fast screening of new chemical molecules which may help against oxidative stress in RBCs, and thereby have tremendous scope in medical diagnostics and therapeutics.