Abstract
BackgroundIn large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA) for quantifying insulin from dried blood spots and demonstrate its application in a large trial.MethodsWe adapted an existing commercial kit (Mercodia Human Insulin ELISA, 10-1113-01) to quantify insulin from two 3-mm diameter discs (≈6 µL of blood) punched from whole blood standards and from trial samples. Paediatricians collected dried blood spots in a follow-up of 13,879 fasted children aged 11.5 years (interquartile range 11.3–11.8 years) from 31 trial sites across Belarus. We quantified bloodspot insulin levels and examined their distribution by demography and anthropometry.ResultsMean intra-assay (n = 157) coefficients of variation were 15% and 6% for ‘low’ (6.7 mU/L) and ‘high’ (23.1 mU/L) values, respectively; the respective inter-assay values (n = 33) were 23% and 11%. The intraclass correlation coefficient between 50 paired whole bloodspot versus serum samples, collected simultaneously, was 0.90 (95% confidence interval 0.85 to 0.95). Bloodspot insulin was stable for at least 31 months at −80°C, for one week at +30°C and following four freeze-thaw cycles. Paediatricians collected a median of 8 blood spots from 13,487 (97%) children. The geometric mean insulin (log standard deviation) concentrations amongst 12,812 children were 3.0 mU/L (1.1) in boys and 4.0 mU/L (1.0) in girls and were positively associated with pubertal stage, measures of central and peripheral adiposity, height and fasting glucose.ConclusionsOur simple and convenient bloodspot assay is suitable for the measurement of insulin in very small volumes of blood collected on filter paper cards and can be applied to large-scale epidemiology studies of the early-life determinants of circulating insulin.
Highlights
We describe the development and validation of an enzyme-linked immunoassay (ELISA) for quantification of insulin from dried blood spots using a simple adaptation of an existing commercial kit originally designed for use on serum or plasma (Mercodia Human Insulin ELISA, catalogue number: 10-1113-01, Mercodia AB, Sweden)
We show the successful practical application of our dried blood spot assay in a large-scale, multicentre trial by describing the distribution and correlates of fasting insulin levels measured on over 12,000 children aged 11.5 years from 31 polyclinics distributed across the Republic of Belarus
[36] The Human Insulin ELISA kit was from Mercodia AB, Sweden, which has no detectable cross-reactivity against c-peptide or proinsulin and showed excellent agreement against an isotope dilution–liquid chromatography/tandem mass spectrometry (IDMS) measurement procedure calibrated using purified recombinant insulin
Summary
Raised insulin levels in children are a marker for insulin resistance, [1,2] elevated cardiovascular disease risk factors[3,4,5,6] and early atherosclerosis. [7] high insulin levels in childhood track into adulthood, [8,9] when they are associated with type 2 diabetes,[10,11,12] cardiovascular disease[13,14,15,16] and premature mortality. [16,17] There is, substantial interest in large-scale epidemiology studies of the genetic and environmental determinants of insulin levels in childhood[18,19,20,21,22,23,24,25,26,27] to inform strategies for the prevention of insulin resistance and its sequelae. [28].Important challenges in large-scale epidemiology include nonacceptance of venepuncture by children and/or their parents; the costs, safety and logistics of serum or plasma separation by centrifugation; and frozen storage and transport of aliquots. [29,30] The major advantages of dried blood spot sampling are minimal training, lower cost than venepuncture, acceptability to parents and children, [30] negligible processing requirements (cards must be air dried), low biohazard risk because samples cannot leak and the ease of storage and transport of filter paper cards. There is, interest in developing and validating a wide range of bloodspot assays, and developments include radioimmunoassay [32] and chemiluminescence [33] methods for insulin quantification in dried blood spots. Development and validation of an ELISA blood spot assay offers a simple, convenient and novel alternative method of measuring insulin in large-scale epidemiology studies in a wide range of settings. In large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA) for quantifying insulin from dried blood spots and demonstrate its application in a large trial
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