Estimation of the Volume of Blood in a Small Disc Punched From a Dried Blood Spot Card

A specialized dried blood spot (DBS) collection system (PUFAcoat) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has enabled the measurement of numerous analytes in minimal volumes of blood. The current study aimed to determine the volume of blood in 3 and 6mm discs obtained from our DBS system. The volume of blood in 3 and 6mm discs obtained from DBS cards is estimated using four different methods: (i) gravimetric analysis; (ii) LC-MS/MS; (iii) a hemoglobin colorimetric assay; and (iv) GC. Differences in the estimated volume are compared between methods, and variations in estimated blood volume within and between individuals are determined. The average volume of blood in a DBS disc is calculated to be 1.60.4L and 8.71.9L for 3 and 6mm discs, respectively. This estimate is similar between direct and indirect analytical methods and between DBS samples with different starting volumes independent of the method, but there is considerable variation in the volume of blood in comparably-sized DBS discs from different individuals. Current methods enable the estimation of the blood volume in a small disc obtained from a DBS but a method that can both accurately measure volume and store blood on a DBS is required.Practical Applications: This study demonstrates that it is possible to evaluate the volume of blood contained in a small disc punched from a dried blood spot (DBS) card using a range of different methods, but there is considerable variation in the estimate of volume in samples collected from different individuals. These variations could potentially result in overestimation or underestimation of endogenous levels of various metabolites if presuming a fixed volume, which may be clinically significant. Although there are several ways to correct for blood volume contained on a DBS, the practicality and the universality of these methods are questionable. Ideally, the development of a tool to better determine the volume of blood and/or to precisely spot a volume of blood is required to ensure accuracy when expressing the results of DBS analyses per unit of blood. The volume of blood in a 3 or 6mm disc obtained from a DBS needs to be known to accurately quantify endogenous levels of metabolites per unit of blood using liquid chromatography-tandem mass spectrometry.