Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measured by tandem mass spectrometry

Background: Detection of amino acids (AA), acylcarnitines (AC), and guanidinoacetate (GAA) in dried blood spots by tandem mass spectrometry has made it possible to detect different inborn errors of metabolism in neonatal screening programs. Despite its proven sensitivity many issues related to sample preparation remain unsolved. Hematocrit has a profound effect on blood viscosity, and may thereby influence flux and diffusion properties of the blood. As newborn infants show a considerable interindividual variability of hematocrit levels, we investigated its effect on levels of AA and AC in dried blood spots. Methods: Blood samples with defined hematocrit levels (20%, 30%, 40%, 50%, 60%) were produced by diluting blood cells with plasma from a single donor. Forty dried blood spots were made for each hematocrit level and a central as well as a peripheral 3 mm disk was punched and analysed for AA, AC, and GAA, respectively. Results: Levels of most AA and GAA increased significantly with increasing hematocrit (p < 0.001), while the effect of hematocrit on some AA was less pronounced. Total AC, free carnitine, some long, medium and short chain AC correlated positively with hematocrit levels (p < 0.001). In samples with low hematocrit, levels of most AA and free carnitine were higher in the peripheral than in the central disk (p < 0.0001). Conclusions: Both hematocrit and position of the disk within the dried blood spot have a significant and sometimes additive effect on levels of AA, AC and GAA in dried blood spots. Theoretically, diagnoses may be missed depending on hematocrit and position of the disk.