Correlation between blood volume and blood spot area. Increasing volumes (1 μl – 45 μl) of fresh heparinized blood were pipetted (five replicates per volume) onto Whatman 3 MM, Whatman No 1 or glass fibre filter paper, dried at ambient temperature and RH and optically scanned. Spot areas were measured by integration and plotted against the volume of blood applied. a) The relationship between volume and blood spot is linear over the entire range. A 2.5 mm diameter spot represents 0.77 μl and 1.4 μl of blood for No 1 paper and 3 MM paper respectively. Whatman No 1 filter paper (- -□- -) and Whatman 3 MM filter paper (.....○.......). b) Glass fibre paper. For blood volumes above 10 μl, spot size is linearly related to blood volume with a 2.5 mm diameter spot representing a volume of 2.2 μl of blood. For blood volumes below 10 μl (inset) the relationship between spot size and volume is unreliable.