Cristina Dickie de Castilhos

Logistica de coleta e transporte de material biologico e organizacao do laboratorio central no ELSA-Brasil

The ELSA (Estudo Longitudinal de Saude do Adulto - Brazilian Longitudinal Study for Adult Health) is a multicenter cohort study which aims at the identification of risk factors associated with type 2 diabetes and cardiovascular diseases in the Brazilian population. The paper describes the strategies for the collection, processing, transportation, and quality control of blood and urine tests in the ELSA. The study decided to centralize the tests at one single laboratory. The processing of the samples was performed at the local laboratories, reducing the weight of the material to be transported, and diminishing the costs of transportation to the central laboratory at the Universidade de Sao Paulo Hospital. The study included tests for the evaluation of diabetes, insulin resistance, dyslipidemia, electrolyte abnormalities, thyroid hormones, uric acid, hepatic enzyme abnormalities, inflammation, and total blood cell count. In addition, leukocyte DNA, urine, plasma and serum samples were stored. The central laboratory performed approximately 375,000 tests.

Influence of pre-analytical factors on α-galactosidase A, arylsulfatase B and α-glucosidase activities measured on dried blood spots on filter paper

OBJECTIVES To analyze the effect of blood collection and storage conditions on activity of α-galactosidase A, arylsulfatase B and α-glucosidase. DESIGN AND METHODS Blood was collected in EDTA, heparin, or direct spotting on filter paper and stored at different temperatures (-20, 4, 25 and 37°C) and storage times (3, 10, 17 and 180 days). The influence of filter paper size was also assessed (3.0 and 1.2mm). RESULTS No statistically significant difference was observed between the three collection methods. α-Glucosidase A activity significantly decreased after the 10th day, while arylsulfatase B activity only differed significantly after the 180th day, and α-galactosidase A activity remained constant throughout this storage time. Excellent correlation coefficients were observed for the two filter paper sizes used. CONCLUSIONS Both paper sizes may be employed. Filter paper specimens should be transported under refrigeration as soon as possible after blood collection.

Determination of the lysosomal hydrolase activity in blood collected on filter paper, an alternative to screen high risk populations.

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.

Delineamento e implementacao do biobanco do ELSA-Brasil: estudo prospectivo na populacao brasileira

The Brazilian Longitudinal Study for Adult Health (ELSA-Brasil) is a multicenter prospective cohort of civil servants designed to assess the determinants of chronic diseases, especially cardiovascular diseases and type 2 diabetes. The present article describes the main design and implementation points of the ELSA-Brasil biobank project. Economic, political, logistical and technological aspects of this study are characterized. Additionally, it discusses the final biorepository protocol and the facilities implemented to achieve this objective. The design and implementation process of the ELSA-Brasil biobank took three years to be performed. Both the central and local biobanks were built according to the best biorepository techniques, using different technological solutions for the distinct needs expected in this study.

Screening of high-risk Gaucher disease patients in Brazil using miniaturized dried blood spots and leukocyte techniques.

This study investigates the miniaturization of the screening technique using dried blood spots on filter paper (DBS) to measure GBA and CT activities, and GBA and β-galactosidase activities in leukocytes. 274 DBS from individuals with suspected GD were screened for 1.5 years. Of these, we confirmed the diagnosis in 13.5%. The miniaturization of the DBS and leukocyte techniques afforded to reduce costs and sample size appropriate for a reliable diagnosis.

Effect of sample collection, temperature and time of storage on β-galactosidase and total hexosaminidase activities in dried blood collected on filter paper

Abstract Background: Dried blood spots (DBS) on filter paper is a valuable sampling technique in clinical chemistry, but the stability of enzymes used in the diagnosis of lysosomal storage diseases (LSDs) needs to be evaluated. Methods: In a first experiment, blood from 20 subjects was collected using a syringe without additives and distributed into EDTA tubes, heparin tubes, and spotted on filter paper for the comparison of sampling effects. In a second experiment, blood from 30 healthy subjects was spotted on filter paper and analyzed for β-galactosidase and total hexosaminidase activities after storage of the samples at different temperatures for up to 180 days. Results: Initially, we observed that enzyme activities were the same, independent of the collection method. When DBS was stored at 37°C the activity of β-galactosidase dropped to 85% of the initial value after 180 days (p<0.05). At all other temperatures (–20°C, 4°C and 25°C), the results were within the methodological error. Total hexosaminidase activity did not change significantly during the entire study period and at different storage temperatures. Conclusions: The two enzymes investigated in the present study may be stored for up to 17 days (β-galactosidase) or 180 days (total hexosaminidase) until analysis without loss of activity.

Application of a protocol for the detection of disorders of sialic acid metabolism to 124 high-risk Brazilian patients

Lysosomal storage disorders (LSD) present great clinical variability. Included in this group are sialic acid metabolism disorders (SAMD). In the present study, we describe the application of a 3‐step protocol for the diagnosis of SAMD, including (1) oligosaccharide and sialyloligosaccharide chromatography; (2) quantitative determination of sialic acid; and (3) measurement of neuraminidase activity. Application of our protocol to 124 individuals at risk for SAMD led to the diagnosis of five affected patients, two with type I sialidosis, one with type II sialidosis, and two with galactosialidosis. Due to its simplicity and efficiency, we propose the use of this protocol for the diagnostic evaluation of patients with suspected SAMD, which could be specially useful to non‐specialized laboratories and to services located in developing countries.

Variability in baseline laboratory measurements of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)

Multi-center epidemiological studies must ascertain that their measurements are accurate and reliable. For laboratory measurements, reliability can be assessed through investigation of reproducibility of measurements in the same individual. In this paper, we present results from the quality control analysis of the baseline laboratory measurements from the ELSA-Brasil study. The study enrolled 15,105 civil servants at 6 research centers in 3 regions of Brazil between 2008–2010, with multiple biochemical analytes being measured at a central laboratory. Quality control was ascertained through standard laboratory evaluation of intra- and inter-assay variability and test-retest analysis in a subset of randomly chosen participants. An additional sample of urine or blood was collected from these participants, and these samples were handled in the same manner as the original ones, locally and at the central laboratory. Reliability was assessed with the intraclass correlation coefficient (ICC), estimated through a random effects model. Coefficients of variation (CV) and Bland-Altman plots were additionally used to assess measurement variability. Laboratory intra and inter-assay CVs varied from 0.86% to 7.77%. From test-retest analyses, the ICCs were high for the majority of the analytes. Notably lower ICCs were observed for serum sodium (ICC=0.50; 95%CI=0.31–0.65) and serum potassium (ICC=0.73; 95%CI=0.60–0.83), due to the small biological range of these analytes. The CVs ranged from 1 to 14%. The Bland-Altman plots confirmed these results. The quality control analyses showed that the collection, processing and measurement protocols utilized in the ELSA-Brasil produced reliable biochemical measurements.

Non-HDL-C goals based on the distribution of population percentiles in ELSA-Brasil: Is it time to change?

BACKGROUND AND AIMS Non-high-density lipoprotein cholesterol (non-HDL-C) goals are defined as 30 mg/dL (0.78 mmol/L) higher than the respective low-density lipoprotein cholesterol (LDL-C) goals. This definition, however, do not consider the population distribution of non-HDL-C, which could represent a more appropriate individual goal when both markers are discordant. The aim of this study is to establish non-HDL-C goals at the same population percentiles of LDL-C. METHODS Non-HDL-C values were assigned at the same percentiles correspondent to the LDL-C treatment goals for 14,837 participants from the Longitudinal Study of Adult Health (ELSA-Brasil) with triglycerides levels ≤ 400 mg/dL (4.52 mmol/L). We also assessed the frequency of reclassification, defined as the number of subjects with LDL-C levels in the recommended therapeutic category, but with non-HDL-C levels above or below the category. RESULTS The non-HDL-C values, based on correspondent LDL-C population percentiles, were 92 (2.38), 122 (3.16), 156 (4.04), 191 (4.95), and 223 mg/dL (5.78 mmol/L). Among participants with LDL-C <70 mg/dL (1.81 mmol/L), 22.8% were reclassified in a higher category according to the guidelines-based non-HDL-C cut-off and 30.1% according to the population percentile-based cut-off; 25.6% and 64.1%, respectively, if triglycerides concurrently 150-199 mg/dL (1.69-2.25 mmol/L). CONCLUSIONS Our results demonstrated that non-HDL-C percentiles-based goals were up to 8 mg/dL (0.21 mmol/L) lower than the guidelines recommended goal and had a profound impact on the reclassification of participants, notably when LDL-C was <100 mg/dL (2.56 mmol/L), the treatment goal for high risk patients. Therefore, non-HDL-C goals should be changed for reduction of residual risk.