Filter paper blood spot enzyme linked immunoassay for adiponectin and application in the evaluation of determinants of child insulin sensitivity

Abstract

Background: Adiponectin is an adipocyte-derived hormone that acts as a marker of insulin sensitivity. Bloodspot sampling by fingerstick onto filter paper may increase the feasibility of large-scale studies of the determinants of insulin sensitivity. We first describe the validation of an enzyme-linked immunoassay (ELISA) for quantifying adiponectin from dried blood spots and then demonstrate its application in a large trial (PROBIT).

Methods: We quantified adiponectin from 3-mm diameter discs (≈3 µL of blood) punched from dried blood spots obtained from: i) whole blood standards (validation); and ii) PROBIT trial samples (application) in which paediatricians collected blood spots from 13,879 children aged 11.5 years from 31 sites across Belarus. We examined the distribution of bloodspot adiponectin by demographic and anthropometric factors, fasting insulin and glucose.

Results: In the validation study, mean intra-assay coefficients of variation (n=162) were 15%, 13% and 10% for 'low' (6.78 µg/ml), 'medium' (18.18 µg/ml) and 'high' (33.13 µg/ml) internal quality control (IQC) samples, respectively; the respective inter-assay values (n=40) were 23%, 21% and 14%. The correlation coefficient between 50 paired whole bloodspot versus plasma samples, collected simultaneously, was 0.87 (95% CI: 0.78 to 0.93). Recovery of known quantities of adiponectin (between 4.5 to 36 µg/ml) was 100.3-133%. Bloodspot adiponectin was stable for at least 30 months at -80°C. In PROBIT, we successfully quantified fasting adiponectin from dried blood spots in 13,329 of 13,879 (96%) children. Mean adiponectin (standard deviation) concentrations were 17.34 µg/ml (7.54) in boys and 18.41 µg/ml (7.92) in girls and were inversely associated with body mass index, fat mass, triceps and subscapular skin-fold thickness, waist circumference, height and fasting glucose.

Conclusions: Bloodspot ELISA is suitable for measuring adiponectin in very small volumes of blood collected on filter paper and can be applied to large-scale studies.

Figure 1. Typical calibration curve.

Whole bloodspot adiponectin standards at 3.75, 7.5, 15.0, 30.46 µg/ml.

Figure 2. Stability of adiponectin concentrations (at A. low, B. medium and C. high values) measured from dried blood spots and stored at −80°C for 30 months.

Adiponectin concentrations of internal quality control (IQC) bloodspots (low, medium and high values) analysed at regular intervals over 30 months. For the ‘low’ IQC sample (Fig. 2A), the middle red line is the mean adiponectin concentration and the upper and lower red lines are the 95% reference range (calculated from the standard deviation of 24 replicates); the respective lines showing the mean and 95% reference range for the ‘medium’ (Fig. 2B) and ‘high’ (Fig. 2C) IQC samples are also shown.

Figure 3. Comparison of adiponectin concentrations measured from 50 paired plasma and whole bloodspot samples.

Figure 4. Bland-Altman plot for plasma adiponectin minus bloodspot adiponectin values in 50 paired samples.

Limits of agreement (reference range for the difference): −14.8 to 0.68 µg/ml. Mean difference in adiponectin between plasma minus blood spot samples: −7.1 µg/ml (95% CI: −8.2 to −6.0). Range of average values: 6.7 to 39.8 µg/ml.

Figure 5. Number of blood spots per child collected in the PROBIT study when the children were aged 11.5 years.