Abstract
To establish a method that allows for the reliable assessment of vascular endothelial growth factor (VEGF-A) concentrations in very small blood samples of preterm infants. Systemic VEGF measurements are important in view of the most appropriate Anti-VEGF drug to be used for the treatment of acute retinopathy of prematurity (ROP). Cord blood samples from preterm (n = 6) infants, blood samples from preterm infants with treatment requiring ROP (n = 12), and blood samples from healthy adults (n = 10) were collected. Serum, citrate plasma, and serum from recalcified citrate blood were obtained. Levels of VEGF-A and platelet factor-4 (PF-4) were quantified by ELISA or AlphaLISA immunoassay. VEGF-A levels could be detected by both assays, with the AlphaLISA generating slightly lower levels in healthy adults, but not in cord blood of preterm infants. In plasma samples, VEGF levels ranged from non detectable to 181 pg/ml. PF-4 concentrations were between 0.16–3.88 µg/ml. Values of VEGF-A and PF-4 in serum and recalcified serum were significantly higher compared to plasma through the release of these cytokines after platelet activation. In plasma samples of infants with ROP, VEGF-A could always be detected and its values ranged from 19.50 to 245.91 pg/ml and PF-4 concentrations were between 0.1 and 3.3 µg/ml. Using the AlphaLISA kit, we were able to detect VEGF in small sample volumes (5 µl plasma or serum/well) in premature infants with treatment requiring ROP and to monitor platelet activation by PF-4 detection. Minimal blood probe volumes reduce phlebotomy losses avoiding the risk of iatrogenic anemia, thus allowing close monitoring of the cytokine levels in these very fragile infants.
Highlights
26–51 years [8,9,10] ml WB, CB associated with higher needs for rescue and recurrence therapies
This is because VEGF-A, together with other growth factors such as platelet factor-4 (PF-4), angiopoietin-1 (ANG-1), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), clotting proteins, or cytokines, is stored in platelet α-granules and becomes released upon activation[17,18]
The VEGF-A values in plasma from citrated blood determined by ELISA or AlphaLISA assays ranged from below detection level to 181 pg/ml
Summary
26–51 years [8,9,10] ml WB, CB associated with higher needs for rescue and recurrence therapies. VEGF-A levels represent the free circulating form of the cytokine, whereas in serum, an additional pool of platelet-derived VEGF-A contributes to the overall cytokine concentration. This is because VEGF-A, together with other growth factors such as platelet factor-4 (PF-4), angiopoietin-1 (ANG-1), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), clotting proteins, or cytokines, is stored in platelet α-granules and becomes released upon activation[17,18]. With regard to preterm infants, the fragile situation in these patients and the small volume of blood pose additional hurdles for reliable testing procedures and need to be addressed in order to gain further insight into the circulating cytokine levels. In order to verify any release of VEGF-A from platelet α-granules, the levels of PF-4 were quantified as well
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