Whole Blood Transcriptomic Profiling

Whole blood (2.5 mL) was collected in PAXgene Blood RNA tubes (PreAnalytics, Hombrechtikon, Switzerland) on the day of inclusion, and subsequently aliquoted and stored at −80 °C until analysis. To perform target gene amplification and generate IMX-SEV-3b classifier results, the samples were shipped on dry ice to the Inflammatix laboratory (Burlingame, CA, USA) in batches, following the procedure previously described [29 (link)]. RNA was extracted from PAXgene tubes using the RNeasy^® Plus Micro Kit (QIAGEN, Hilden, Germany) on the QiaCube^® Connect instrument. The NanoString nCounter^® SPRINT profiler (NanoString, Seattle, WA, USA) was then used to quantify the 29 host target mRNAs, as well as four housekeeping genes for normalization (CDIPT, KPNA6, RREB1 and YWHAB) from 150 ng of isolated RNA. RNA concentrations were determined for 29 genes of various aspects of the immune response and included CEACAM1, ZDHHC19, C9orf95, GNA15, BATF, C3AR1, KIAA1370, TGFBI, MTCH1, RPGRIP1, HLA-DPB1, HK3, TNIP1, GPAA1, CTSB, IFI27, JUP, LAX1, DEFA4, CD163, RGS1, PER1, HIF1A, SEPP1, C11orf74, CIT, LY86, TST and KCNJ2 [29 (link)]. Operators at Inflammatix were blinded to all other study results, and the NanoString data were subjected to direct application of the SEV-3b classifier, blinded to clinical outcomes.