Validity of Luminex and Enzyme-Linked Immunosorbent Assay Measuring Vascular Endothelial Growth Factor and Six Cytokines Quantitatively in Bone Marrow Aspiration Supernatant: Pilot Study

Abstract

Objective Vascular endothelial growth factor (VEGF) and other cytokines have been reported to be implicated in the molecular pathogenesis of hematologic malignancy. However, a quantitative measurement of VEGF and related cytokines is necessary to reflect the real situation in the bone marrow (BM). Currently, no such quantitative assays exist for use in the BM supernatant as their concentrations have not been previously validated in the BM. Here we performed linearity and recovery tests to quantitatively measure the concentrations of VEGF and six related cytokines in the BM.

Method A total of 24 BM supernatant samples were collected from patients who underwent a BM examination for hematological malignancies. The levels of VEGF and six cytokines - granulocyte colony-stimulating factor (G-CSF), interferon-β (INF-β), interleukin (IL)-1β, IL-6, IL-17A, and tumor necrosis factor-α (TNF-α) - were measured using Luminex assay and enzyme-linked immunosorbent assay. Percentage recovery and linearity were calculated, with the acceptable range being 80-120%. The undiluted and diluted (1:2, 1:4, and 1:8) concentrations of VEGF and the six cytokines in 24 spiked and unspiked BM supernatant samples and controls were also measured.

Results For VEGF, both assays passed the percentage recovery and linearity tests; wherein the undiluted and all diluted concentrations of VEGF in all six unspiked BM samples showed linearity parallel to those of VEGF in spiked BM samples and controls. For the other six cytokines, both assays did not pass the percentage recovery and linearity tests, with the undiluted and diluted concentrations in all seventeen unspiked BM samples (except G-CSF in one sample) showing a lack of parallelism to those in spiked BM samples and controls.

Conclusions Quantitative VEGF measurement in real BM specimens was validated using both Luminex assay and ELISA. All six cytokines, except for VEGF, whether undiluted or diluted, could not be accurately measured in the BM supernatants, indicating the presence of inhibitors to the analytes. Quantitative measurement of VEGF-related cytokines in the BM will have to be validated in further studies with more samples.