Filter paper blood spot enzyme linked immunoassay for adiponectin and application in the evaluation of determinants of child insulin sensitivity.
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.
School of Social and Community Medicine, University of Bristol, Bristol, United Kingdom. richard.martin@bristol.ac.uk
Richard M
RM
Martin
Rita
R
Patel
Emily
E
Oken
Jennifer
J
Thompson
Alexander
A
Zinovik
Michael S
MS
Kramer
Konstantin
K
Vilchuck
Natalia
N
Bogdanovich
Natalia
N
Sergeichick
Ying
Y
Foo
Nina
N
Gusina
D052242:Adiponectin / Q000097:blood*; D000293:Adolescent; D000328:Adult; D002648:Child; D059788:Dried Blood Spot Testing / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005374:Filtration; D006801:Humans; D007333:Insulin Resistance; D008297:Male; D010209:Paper; D015203:Reproducibility of Results
D052242:Adiponectin