A systematic review and meta-analysis of preanalytical factors and methodological differences influencing the measurement of circulating vascular endothelial growth factor
Authors: Sjöbom Ulrika , Nilsson Anders K. , Gyllensten Hanna , Hellström Ann , Löfqvist Chatarina 
Published: July 2022

Background
Intraocular treatment with antibodies targeting vascular endothelial growth factor (anti-VEGF) inhibits pathological vessel growth in adults and preterm infants. Recently, concerns regarding the impact of anti-VEGF treatment on systemic VEGF levels in preterm infants have been raised. Earlier studies suggest that preanalytical and methodological parameters impact analytical VEGF concentrations, but we have not found a comprehensive systematic review covering preanalytical procedures and methods for VEGF measurements.

Objective
This review aimed to evaluate the most critical factors during sample collection, sample handling, and the analytical methods that influence VEGF levels and therefore should be considered when planning a prospective collection of samples to get reproducible, comparable results.

Material and methods
PubMed and Scopus databases were searched 2021/Nov/11. In addition, identification of records via other methods included reference, citation, and Google Scholar searches. Rayyan QCRI was used to handle duplicates and the selection process. Publications reporting preanalytical handling and/or methodological comparisons using human blood samples were included. Exclusion criteria were biological, environmental, genetic, or physiological factors affecting VEGF. The data extraction sheets included bias assessment using the QUADAS-2 tool, evaluating patient selection, index-test, reference standard, and flow and timing. Concentrations of VEGF and results from statistical comparisons of analytical methods and/or preanalytical sample handling and/or different sample systems were extracted. The publications covering preanalytical procedures were further categorized based on the stage of the preanalytical procedure. Meta-analysis was used to visualize VEGF concentrations among healthy individuals. The quality of evidence was rated according to GRADE.

Results
We identified 1596 publications, and, after the screening process, 43 were considered eligible for this systematic review. The risk of bias estimation was difficult for 2/4 domains due to non-reported information. Four critical steps in the preanalytical process that impacted VEGF quantification were identified: blood drawing and the handling before, during, and after centrifugation. Sub-categorization of those elements resulted in nine findings, rated from moderate to very low evidence grade. The choice of sample system was the most reported factor. VEGF levels (mean [95% CI]) in serum (n = 906, 20 publications), (252.5 [213.1–291.9] pg/mL), were approximated to ninefold higher than in plasma (n = 1122, 23 publications), (27.8 [23.6–32.1] pg/mL), based on summarized VEGF levels with meta-analysis. Notably, most reported plasma levels were below the calibration range of the used method.

Conclusion
When measuring circulating VEGF levels, choice of sample system and sample handling are important factors to consider for ensuring high reproducibility and allowing study comparisons. Protocol: CRD42020192433