Abstract Number: PB0443
Meeting: ISTH 2020 Congress
Background: With the large implication of NET in physiopathological processes and intensive use of biomarkers of NET in clinical fields, preanalytic standardization is required.
1) Evaluate 3 types of blood sampling (serum, plasma citrate and EDTA) on DNA quantitation, MPO-DNA complex and DNase activity.
2) Evaluate the stability of these parameters in citrated plasma at 2, 6 and 24h after blood collection.
– Cell-free DNA was quantified using Quant-iT PicoGreen® dsDNA Reagent and MPO-DNA complex according a modified approach of the Cell-Death Detection Elisa kit. For plasmatic DNase activity we used a fluorometric determination in microplates containing salmon-DNA as substrate and recombinant human DNaseI as calibration.
– Preparation and storage of serum, plasma citrate and EDTA samples was carried out in accordance with the French guideline procedures.
– For Aim 1 we used blood from 10 healthy controls and for aim 2, 9 healthy volunteers and 9 patients with myeloproliferative neoplasms.
– Aim 1: Cell free-DNA levels were significantly higher in serum. MPO-DNA quantification was not significantly different whatever the type of blood sampling; nevertheless with EDTA, the values were more dispersed. In citrated plasma we noted a significant increase of DNase activity compared to sera or EDTA plasma. This could be due a better preservation of DNase activity in citrate, due to lower deprivation of divalent ions.
– Aim 2: No significant variation was observed for DNA levels and DNase activity at the different time points. For MPO-DNA, we observed significant differences between 2-6h in citrate.
Conclusions: As highlighted by Meddeb in 2019, standardizing preanalytical conditions is required to compare results for clinical research. Based on our results, citrate blood sampling seems to be more relevant for a simultaneous analysis of biomarkers of NETosis. To maintain analyte integrity, sampling should be carried out no more than two hours after blood collection.
To cite this abstract in AMA style:Labrouche-Colomer S, Guy A, Jeanningros S, James C. Preanalytical Considerations for Evaluation of NETosis Biomarkers [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/preanalytical-considerations-for-evaluation-of-netosis-biomarkers/. Accessed October 1, 2023.
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