Diagnóstico laboratorial de hemoglobinopatias em populações diferenciadas

The inherited haemoglobinopathies are a heterogeneous group of recessive disorders that include the thalassaemias and sickle cell disease. Nearly a thousand mutant alleles have now been characterized. The mutations are regionally specific and in most cases the geographical and ethnic distribution shave been determined providing the foundation for a program of control through screening, genetic counseling and prenatal diagnosis. The diagnosis of hemoglobinopathies requires care for the methodologies applied and the population group which will be evaluated. The information about the abnormal hemoglobin, the medical and psychological aspects and genetic counseling of the carriers and their families are goals of great importance for the success of preventive programs in this area. Aiming to evaluate the laboratory methods for hemoglobinopathy screening and their use in clinical laboratories, we have compared abnormal hemoglobins incidence in the different population groups: blood donors, anemia carriers, newborn and students. The laboratory methods applied involved eletrophoretic proceedings, cytological and biochemical analysis. Within the period from September 1999 through January 2000, we analyzed 524 individuals with varied types of abnormal hemoglobins. Among blood donors, we diagnosed two sickle cell carriers, which suggest the necessity for better care in the process of selection of blood donor candidates. The current interest in the medical and social aspects of sickle cell anemia has resulted in a great increase in methodology research leading to the development of sickle cell screening techniques.

Hemoglobin as AS/alfa talassemia - Importância diagnóstica

Sickle Cell disease is a generic term for a group of genetic disorders characterized by the predominance of hemoglobin S. These disorders include Sickle Cell anemia, the Sickle Cell beta Thalassemia syndromes and Hemoglobinopathies in which hemoglobin S is in association with another abnormal hemoglobin, such as hemoglobin S/C. The Sickle Cell trait (hemoglobin AS) associated with Alpha Thalassemia presents alterations in the red blood cells morphology, usually absent in the heterozygous for this hemoglobin variant. The interaction between hemoglobin Sand alpha Thalassemia has been described as one of the factors responsible for the improvement in the clinical picture of homozygous of hemoglobin S (Sickle Cell Anemia), decreasing the number of episodes of pain. The genetic mechanisms of this influence are evaluated using molecular analyses of the human globin genes. With the objective of verifying the presence of alpha Thalassemia in heterozygous of hemoglobin S, with anemia, sent to the Laboratory of Hemoglobins, Department of Biology, UNESP, São José do Rio Preto, SP, we analyzed 1002 blood samples with Sickle Cell trait, in the period from 1990 to 1998. The samples were picked with EDTA 5% as anticoagulant, after previous authorization of the carriers. Appropriated counseling and management requires definitive diagnosis. For the laboratorial diagnosis the blood samples were submitted to electrophoretic procedures in alkaline and acid pH and cytological evaluation of hemoglobin H. The electrophoretic procedures confirmed the presence of hemoglobin AS. The cytological evaluation evidenced the presence of alpha Thalassemia. Of this total analyzed, 16(1,59%) blood samples presented the association between hemoglobin AS and alpha Thalassemia and two individuals belonged of the same family. Our results addressed us to suggest to the routine laboratories, that is important to accomplish the research of alpha Thalassemia among the Sickle Cell trait, with anemia, to verify the interaction with alpha Thalassemia, supplying to the carriers a important information on its hematological profile, genetic pattern of hemoglobinopathies and the appropriated counseling. Rev.bras.hematol.hemoter.,2000,22(3):388-394.

Uso do óxido nítrico em pediatria

Objective: To review the literature on inhaled nitric oxide and to describe its main clinical applications in pediatrics. Sources of data: A 10 year literature review with selection of the most important publications on inhaled nitric oxide, using the Medline and Cochrane Systematic Review databases. Summary of the findings: This review was organized as follows: introduction; metabolism and biological effects; clinical applications; dosage, gas administration and weaning; precautions and side-effects. Inhaled nitric oxide use was described in persistent pulmonary hypertension and hypoxia of the newborn, acute respiratory distress syndrome, primary pulmonary hypertension, heart surgery, chronic obstructive pulmonary disease, sickle cell anemia, and bronchospastic disease. Conclusions: Inhaled nitric oxide is a therapeutic approach with wide clinical applications in pediatrics. Its use is safe when administered in pediatric intensive care units under strict monitoring. As a pulmonary vasodilator, nitric oxide has beneficial effects on gas exchange and ventilation. Controlled trials, focusing on early gas administration should be performed under many clinical conditions, especially acute respiratory distress syndrome.

Standardization of platelet parameters determination in blood samples collected with EDTA

The Abbott Cell-Dyn 3000 automated hematological analyzer prepares a histogram of platelet volume, which is measured by a technique using electrical impedance, inferring from its platelet count (PLT), mean platelet volume (MPV) and platelet distribution width (PDW). This equipment also calculates the plateletcrit (PCT). These platelet parameters may be important to evaluate platelet function, but they require standardization, because platelets swell when in contact with ethylenediaminetetra-acetic acid salts, hence an increase in blood sample storage time produces artificially increased results. To assess the effect of storage time on MPV, PLT, PDW and PCT, blood samples from 23 sickle cell anemia patients during steady state (Group I) and 50 from healthy controls (Group II) were placed in Vacutainer® tubes with dipotassium ethylenediaminetetra-acetic acid and measured over a period of 1440 minutes (24 h) at the following times: immediately after the venipuncture (time 0), 15, 30, 60, 120, 240, 360, 480 and 1440 minutes. The mean values of MPV and PCT were significantly increased (p<0.00001) along the storage time in both groups. The mean values of PLT and PDW were practically stable (p>0.05) throughout the storage time in both groups.

Microondas doméstico na síntese de derivados ftalimídicos

Microwaves have been used in organic synthesis, since 1986, and have proved advantageous in several respects: the possibility of higher yields, greater selectivity and less thermal decomposition. Phthalimide and its derivatives constitute an important class of compounds for use in synthetic organic chemistry; in medicinal chemistry, it is considered an important biophore, acting as a pharmacophoric structural subunit for the synthesis of a number of compounds with different pharmacological uses, such as against sickle-cell disease. The purpose of the work reported here was to develop an alternative method for the synthesis of phthalimide derivatives by exploiting the condensation of phthalic anhydride with amino groups under microwave radiation. The results showed that phthalimide derivatives were obtained in shorter reaction times (5-10 min) and higher yields (60-89%) than by with conventional heating (reflux), demonstrating the potential use of microwaves in the synthesis of this class of molecules.

Análise das prevalências de doenças detectadas pelo programa nacional de triagem neonatal no município de Araraquara no ano de 2009

The National Neonatal Screening Program (NNSP) set up in all Brazil, aims, through planned phases of local implementation, to detect diseases such as phenylketonuria, congenital hypothyroidism, hemoglobinopathies and cystic fibrosis. The aim of this study was to assess, through a cross-sectional observational study, the prevalence of the diseases detected by the NNSP in the city of Araraquara, in records issued by the São Paulo APAE laboratory in the period between April and December 2009.The results show that Araraquara had a prevalence of phenylketonuria and congenital hypothyroidism 0.06% above the national averages of 0.01% and 0.03% respectively. With respect to hemoglobinopathies, the prevalence of sickle cell trait was 2.15% below the national average of 2.6%. The prevalence of Hb C in the city was 0.57%, similar to national values reported in the literature. Confirmed Hb Bart's had a prevalence of 0.13% in Araraquara, below the average of 0.38% for the surrounding region. The neonatal screening by heel-prick test and counseling for caregivers are important factors in reducing morbidity related to the evolution of these diseases.

Avaliação da correlação entre aloimunização à antígenos eritrocitários e Sistema de histocompatibilidade (HLA) em pacientes com doença falciforme

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Estresse oxidativo em doentes falciformes: influência dos haplótipos e udo de medicação específica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Polimorfismo nos genes Metilenotetrahidrofolato redutase e cistationina-beta-sintase e a sua relação com eventos vaso-oclusivos na doença falciforme

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expressão fenotípica da homozigose para hemoglobina S em relação aos haplótipos da beta globina, polimorfismos da glutationa S-transferase e enzimas de detoxificação

Pós-graduação em Genética - IBILCE

Efeito da Hidroxiuréia, uma substância de uso médico, sobre parâmetros biológicos de Drosophila melanogaster

Pós-graduação em Genética - IBILCE

Determinantes genéticos, bioquímicos e clínicos na resposta ao uso de hidroxiureia na doença falciforme

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of beta(S) allele in the lipid peroxidation and antioxidant capacity parameters

Introduction: The oxidative process plays a fundamental role in the pathophysiology of sickle cell anemia (SCA), and population and environmental characteristics may influence redox balance. The aim of this study was to evaluate lipid peroxidation and antioxidant capacity in Brazilian Hb S carriers undergoing different therapies.MethodsBlood samples from 270 individuals were analyzed (Hb SS, n=68; Hb AS, n=53, and Hb AA, n=149). Hemoglobin genotypes were assessed through cytological, electrophoretic, chromatographic, and molecular methods. Plasma lipid peroxidation and antioxidant capacity were measured by spectrophotometric methods.ResultsPatients with SCA who used iron-chelating drugs combined with hydroxyurea, associated with regular transfusions, showed lower levels of TBARS (P <= 0.05), higher levels of TEAC (P <= 0.01), and lower TBARS/TEAC ratio (R=255.8). The redox profile of Hb AS subjects was not statistically different (P>0.05) from that of Hb AA subjects.ConclusionThe data suggest that oxidative stress is lower in the patients with SCA who received regular blood transfusions associated with the combined use of HU and iron chelators than the group received only HU. The redox system of the Hb AS carriers is compatible with the control group.

Phenotypic expression of homozygous hemoglobin S in relation to β-globin haplotypes, glutathione S-transferase polymorphisms and detoxification enzymes

Sickle cell anemia (SCA) shows a pathophysiology that involves multiple changes in sickle cell erythrocytes, vaso-occlusive episodes, hemolysis, activation of inflammatory mediators, endothelial cell dysfunction, and oxidative stress. These events complicate treatment and culminate in the development of manifestations such as anemia, pain crises and multiorgan dysfunction. The aim of this study was to evaluate, in SCA patients, oxidative stress and antioxidant capacity markers, correlating them to treatment with hydroxyurea (HU), β-globin haplotypes and glutathione S-transferase polymorphisms (GSTT1, GSTM1 and GSTP1), in comparison to a control group (CG). The study groups were composed of 48 individuals without hemoglobinopathies (CG), SCA patients treated with HU [AF (+HU), N = 13] and untreated SCA patients [AF (-HU), N = 15], after informed consent. The groups were analyzed using cytological, electrophoretic, chromatographic and molecular methods and information from medical records. The GSTM1 and GSTT1 polymorphisms were determined by multiplex PCR, while the GSTP1 polymorphism by PCR-RFLP. Biochemical parameters were measured using spectrophotometric methods [TBARS, TEAC and catalase (CAT) and GST activities] and a chromatographic method [glutathione (GSH)]. The fetal Hb (Hb F) levels observed in the SCA (+HU) group (10.9%) confirmed the already well-described pharmacological effect of HU, but the SCA (-HU) group also had high Hb F levels (6.1%), which may have been influenced by genetic factors not targeted in this study. We found a higher frequency of the Bantu haplotype (48.2%), followed by the Benin (32.1%) and also Cameroon haplotypes, rare in our population, and 19.7% of atypical haplotypes. The presence of Bantu haplotype was related to higher lipid peroxidation levels in patients, but also, it conferred a differential response to HU treatment, raising Hb F levels in 52.6% (P = 0.03). The protective effect of Hb F was confirmed, because the increase in their levels resulted in a 41.3% decrease in lipid peroxidation levels (r = -0.74, P = 0.0156). The genotypic frequency of the GST polymorphisms observed was similar to that of other studies in the Brazilian population, and its association with biochemical markers revealed a significant difference only for the GSTP1 polymorphism, where patients with genotype V/V showed higher GSH and TEAC levels (P = 0.04 and P = 0.03, respectively) compared to patients with genotype I/I. The TBARS levels were about five to eight times higher in the SCA (+HU) and SCA (-HU) groups, respectively, compared to controls, and HU produced a 35.2% decrease in lipid peroxidation levels in the SCA (+HU) group (P < 0.0001). Moreover, the SCA (+HU) group showed higher TEAC levels when compared to CG (P = 0.002). We did not find any significant difference in GST activity between the groups studied (P = 0.76), but CAT activity was about 17 and 30% lower in SCA (+HU) and SCA (-HU) groups, respectively (P < 0.00001). Plasma GSH levels were ~2 times higher in SCA patients than in the control group (P = 0.0005) and showed a positive correlation with TBARS levels, confirming its antioxidant function. HU treatment contributed to higher CAT activity and TEAC levels and lower lipid peroxidation, and its pharmacological effect showed a “haplotype-dependent” response. These findings may contribute to elucidating the potential of HU in ameliorating oxidative stress in SCA subjects.

Elementos de regulação e microRNAs envolvidos na modulação dos níveis de hemoglobina fetal em indivíduos portadores de beta-hemoglobinopatias

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)