Fig 1.
Overview of sample acquisition procedures and the distribution of samples from blood acquisition centers to measuring centers and central laboratory.
Table 1.
Investigated parameters.
Table 2.
Results of the multiple regression analysis.
Fig 2.
2.1 (a-c) Paired plots (light orange) and box plots (blue) of VEGF-A levels measured at blood taking centers, measuring centers and at the central laboratory (CL). Connecting lines illustrate the measured VEGF-A levels from identical aliquots of samples measured at the acquisition center, the measuring center and the central laboratory; 2.2 Impact of the anticoagulant (EDTA, PECT, CTAD) on measurement of VEGF-A, PF-4 and IGF-1 from plasma. VEGF-A levels measured from PECT (a) and CTAD (b) plasma are much lower than from EDTA plasma. Similar to VEGF-A, PF-4 values are higher in EDTA plasma than in PECT (c) or CTAD (d) plasma, indicating increased thrombolysis in EDTA samples compared to PECT or CTAD. IGF-1 levels are not affected by the choice of anticoagulant (e, f) indicating that the choice of coagulant is not a general confounder for all biomarkers. (g) VEGF-A levels obtained after spiking VEGF-A into PBS containing EDTA, PECT or CTAD yielded comparable results thus confirming that CTAD or PECT do not interfere with the VEGF ELISA assay. It was noted, however, that VEGF stored at -80°C for over one year yields lower recovery rates in all samples, irrespective of the anticagulant used; 2.3 Impact of the kind of centrifuge used for separating plasma from the cell pellet (equivalent settings were applied). VEGF-A (a) and PF-4 (b) levels were higher in some samples from the fixed angle rotor compared to the swing-out rotor. IGF-1 levels (c) were not affected by the type of centrifuge; 2.4 Comparison of measurements obtained with two methods: ELISA vs. multiplex bead array (Luminex). (a) box-plot analysis showing that absolute values differ between the two measurement methods; (b, c) scatter plot and Bland-Altmann plot, however, demonstrate a good correlation of relative measurement results from the two methods; (d, e) VEGF-A protein standards from both the ELISA kit and the Luminex kit were measured in both assays. In either assay, the VEGF-A standard provided with the Luminex kit resulted in higher signals for identical protein concentrations compared to the VEGF-A standard provided with the ELISA kit. This translates in lower measurements of patient sample VEGF-A levels when these are normalized to the Luminex VEGF-A standard as opposed to the ELISA standard (irrespective of the assay used).
Fig 3.
(a) Comparison of the mathematically expected and measured VEGF-A levels after a 1:5 and 1:10 sample dilution. Both ELISA and multiplex bead array measurements measure slightly higher values than mathematically expected after sample dilution; (b) Addition of up to 1.333 pg/ml of recombinant VEGF-A to a plasma sample yields recovery rates of about 80% in all anticoagulants. Exp. = expected value; Meas. = measured value.
Fig 4.
Impact of time before (a,b,c) and after (d-g) centrifugation on measured VEGF-A and PF-4 levels.
In some EDTA samples, VEGF-A (a,c,d) and PF-4 levels (b,f) increased with longer incubation times. This was particularly significant when EDTA samples were stored for longer periods of time before centrifugation (c). In PECT samples (c,e,g), VEGF and PF-4 values were not affected by longer sample storage times.
Fig 5.
(a) Scatter plot for the influence of cannula (neonatal vs. butterfly) on measured VEGF-A plasma levels; (b) Scatter plot for intrapersonal fluctuations in plasma VEGF-A levels over one week; (c) Scatter plot for the impact of filling level of blood collection tubes on the resulting VEGF-A levels.
Location of values near the bisecting line indicates comparable measurement results.