Analytical Validation of a Commercially Available Canine N-Terminal Prohormone Brain Natriuretic Peptide Elisa
In the past few years NT-proBNP has risen to prominence as a marker for cardiac disease in both dogs and humans. A number of studies have been published looking at the utility of the marker to diagnose cardiac disease. Currently the only assay available for the determination of N-Terminal Prohormone Brain Natriuretic Peptide (NT-proBNP) in dogs is the CardioScreenTM NT-proBNP assay (Guildhay Ltd., UK).
The assay has a quoted limit of detection of 42 pmol/L and the inter-assay and intra-assay variations are both around 7-8%. There is, however, no mention of linearity and so the accuracy of the assay with samples significantly higher than the top standard is unknown. The objective of this study was to independently evaluate the performance of the assay and also to determine the validity of the standard curve with samples well above the highest point of the standard curve.
We ran 10 replicates of 14 different samples from both healthy and sick dogs with NT-proBNP concentrations ranging from 28 to 4039 pmol/L to evaluate intra-assay precision and found that with samples greater than 800 pmol/L the coefficient of variation (CV) was 4-8% which is slightly better than the manufacturer's claim, however with samples below 800 pmol/L the precision was poorer, ranging from 12-20%. The 28 pmol/L was exceptionally poor with a CV of 141%, however this sample is below the assay limit of detection which could explain the poor precision.
Inter-assay variation was examined by running 10 replicates of 4 different samples on 3 different kit lots of plates. The samples used ranged in concentration from 365 to 2215 pmol/L. The three samples which were at or higher than 800 pmol/L had an inter-assay CV of around 7.5% while the low sample was again higher with a CV of about 16%.
The linearity of the standard curve was assessed using 6 samples ranging from 2801 to 6246 pmol/L. Each sample was run neat and diluted 1:5 and 1:10. In the case of the highest sample the difference between the neat and diluted values is close to 40% which may indicate that at the very high end the values obtained are not truly accurate and samples should be diluted and re-run. However, the NT-proBNP concentration is far away from the suggested diagnostic cut-off so this may not be a major concern. The lower samples are generally in good agreement between the neat and diluted samples with the diluted sample values being within 20% of the neat value.
We also investigated the effect of changing the incubation time from the recommended 24 hours down to as low as 3 hours and found no significant effect on precision; though the flatness of the standard curve with incubation times below 7 hours means that while the assay can be shortened, using the current kit components, the incubation step should not be shortened below 7 hours.
Overall, the kit is robust and performs according to the manufacturer's specifications.