Figure 1

Analysis of DBS elution conditions. (A) Total protein concentration in elutes obtained from DBS stored at room temperature, at −80 °C and at −80 °C and air dried (a. d.) for 10 minutes before elution. Elution is performed with buffer for 1 hour at 37 °C or overnight (ON) at 4 °C. There is no variation of total protein concentration in elutes obtained from DBS kept at different storage conditions but there is a significant reduction of total protein concentration when the elution is undertaken ON at 4 °C (Bradford measurement, multiple comparisons Kruskal-Wallis test; RT p = 0.03, −80 °C a.d. p = 0.0006, −80 °C p = 0.002); (B) total protein content in elutes according to pH, storage conditions and use of Tween20 or Barb2. Elution with PBS + Tween20 at 7.4 pH of air-dried DBS stored at −80 °C has the best yield and less variability of total protein concentration in elution products (Bradford measurement; multiple comparisons Kruskal-Wallis test, Tween20 RT vs −80 °C p = 0.007, Tween20 vs Barb₂ p = 0.008); (C) no changes in total protein concentration (Mann-Whitney test, p = 0.33) when elution is performed with or without Tween in the buffer (Pierce measurement). (D) Increasing volumes of elution buffer show linearity in total protein concentration in the elutes up to 1/100; linearity is lost for higher elution volumes; (E) total protein concentration (Pierce measurement) in DBS elutes from Healthy Controls (HC), ALS-Fast and ALS-Slow DBS is about 8 to 10 fold lower than total protein concentration measured in matched plasma samples. There are no differences in the DBS protein levels between HC and ALS patients. (F) Significant correlation between Haemoglobin (Hb), Haematocrit (Hct) and total protein concentrations measured in DBS obtained from the same blood samples (p = 0.0019, R² = 0.32 and p = 0.0055, R² = 0.28 respectively; linear regression analysis). DF: dilution factor.