Presymptomatic diagnosis of postoperative infection and sepsis

Published: 4 July 2022| Version 3 | DOI: 10.17632/rhc5s6zj88.3


Blood samples and clinical/laboratory data were collected daily from patients undergoing elective surgery. A clinical adjudication panel identified patients with definite postoperative infection, some of whom developed sepsis. Transcriptomic data via illumina microarraays were acquired from sequential blood samples, collected up to three days prior to the development of clinical signs and symptoms of infection and and from matched patients with an uncomplicated recovery.


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Overall design: Total RNA obtained from human patients undergoing surgery across three days prior to infection diagnosis, or additional organ dysfunction and controls. Extract protocol: Total RNAs were extracted from an aliquot of 1.832 samples containing 400µl whole blood immersed in 1mL RNAlater using a twostep protocol as implemented in the RiboPure™-Blood Kit (Thermo Fisher Scientific, Part Number AM1928). RNAlater was removed and the cells were resuspended in 800µl guanidinium-based lysis solution. After addition of 50µl Sodium Acetate solution, RNA was extracted with 500µl Acid phenol/chloroform. After phase separation, the aqueous phase was recovered and 600µl of 100% ethanol added for binding of the RNA on a glass fiber filter spin column. After 3 wash steps the RNA was eluted in 2x50µl elution solution. The purity and the concentration of the resulting RNA was determined with spectrophotometry on a Nanodrop™ 8000 and the RNA integrity was analyzed on an Agilent Bioanalyzer. The RNA samples were then normalized to 15ng/µl using 96 well UF plates (Qiagen, Hilden, Germany). Label protocol: cRNA was prepared by amplification and labeling using the Illumina® TotalPrep™ RNA Amplification Kit (ThermoFisher) Hyb protocol: Standard Illumina hybridization protocol Scan protocol: Standard Illumina scanning protocol Data processing: The Illumina® Human HT-12v4 beadarrays were preprocessed and background corrected using GenomeStudio™ Software v2011.1 (Illumina®). Normalization via batch correction including normal and exponential convolution model using the Limma package within R. Value definition: Cyclic-loess normalization


Sepsis, Microarray, Healthcare-Associated Infection