Newborn screening blood spot analysis in the UK: influence of spot size, punch location and haematocrit

Objective: In dried blood spot analysis, punch location and variations in applied sample volume and haematocrit can produce different measured concentrations of analytes. We investigated the magnitude of these effects in newborn screening in the UK. Methods: Heparinized blood spiked with thyroid stimulating hormone (TSH), phenylalanine, tyrosine, leucine, methionine, octanoyl carnitine (C8), and immunoreactive trypsinogen (IRT) was spotted onto filter paper: (i) at a constant haematocrit of 50% at various volumes, and (ii) at a range of haematocrits using a constant volume. Subpunches (3.2mm) of the dried blood spots were then analysed. Results: Compared with a central punch from a 50 mu L blood spot with 50% haematocrit, 10 mu L spots can have significantly lower measured concentrations of all analytes, with decreases of 15% or more observed for leucine, methionine, phenylalanine, and tyrosine. Punching at the edge of a spot can increase measured concentrations up to 35%. Higher haematocrit decreased measured TSH and C8 yet increased amino acids and IRT by 15% compared with 50% haematocrit. Lower haematocrits had the opposite effect, but only with higher concentrations of some analytes. Conclusions: Differences in blood spot size, haematocrit and punch location substantially affect measured concentrations for analytes used in the UK newborn screening programme, and this could affect false positive and negative rates. To minimize analytical bias, these variables should be controlled or adjusted for where possible.