{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Michael Carter"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15164"],"description":["We analysed data collected as part of the Biomarkers of Acute Serious Illness in Children (BASIC) study. BASIC was a prospective cohort study that enrolled critically ill children admitted to four PICUs in London and Eastern England, UK, during emergency transport by the Children’s Acute Transport Service (CATS), the regional paediatric critical care retrieval service from 2014–2016. We aimed to identify, and validate, shared features of immune dysfunction (\\\"endotypes\\\") and assess association with clinically important outcomes (primary outcome: ventilator-free days at day 30). Whole blood was sampled into RNA-stabilising tubes (PAXgene, Qiagen, Germany) from recruited patients immediately during paediatric critical care retrieval. Samples were initially stored on ice packs (4ºC) prior to arrival to the admitting PICU, before storage at -80ºC and processing in batches using Illumina Human-HT-12 version 4 Expression BeadChips (Illumina, CA, USA). Statistical analysis was undertaken in R: A Language and Environment for Statistical Computing (version 4.4.1). Microarray data were compiled, log2 transformed and normalised. Of 384 samples assay, 382 met quality control criteria and went forwards for bioinformatics analysis. We used the top 15% (~5000) most variable transcript probes in the dataset before using k-means clustering (Hartigan and Wong algorithm) with two centres to determine BASIC endotype membership for individual children at retrieval to PICU. BASIC endotype membership was internally validated with a train/validation (2/3 training, 1/3 validation) partition of the dataset, and a nested cross-validation implementation of GLMNet. We identified two robust endotypes, BASIC endotype 1 (122, 31.9%, children), and BASIC endotype 2 (260, 68.1%, children), present in children with diverse illnesses and age groups. BASIC endotype 1 membership was associated with 4.1 (95% CI 2.0–6.2) days of increased length of mechanical ventilation and a non-significant association with mortality. BASIC endotype 1 membership was associated with increase naïve and resting memory CD4 T cell proportions, lower neutrophil proportions and lower gene expression sets associated with tumour necrosis factor (TNF)-α, interferon-γ, interferon-α, and interleukin-6/JAK/STAT pathways in comparison with BASIC endotype 2. These BASIC endotypes may enable stratified trials of treatment for immune dysfunction."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Hybridization - Labeled, amplified material (750 ng per array) was hybridized to the Illumina HumanHT-12 V4.0 expression BeadChip according to the manufacturer's instructions (Illumina, Inc., San Diego, CA).","Sample Collection - Participants We analysed data collected as part of the Biomarkers of Acute Serious Illness in Children (BASIC) study19. BASIC was a prospective cohort study that enrolled critically ill children admitted to four PICUs in London and Eastern England, UK, during emergency transport by the Children’s Acute Transport Service (CATS), the regional paediatric critical care retrieval service (Supplementary Figure 1) from 2014–2016. Children aged 0–16 years with an indwelling central venous or arterial catheter were eligible. Infants <36 weeks of corrected gestational age and those with agreed do-not-attempt-resuscitation plans were not recruited. The study was approved by the Health Research Authority (HRA, reference: 136866) and ethical approval was provided by the National Research Ethics Committee East Midlands—Nottingham 2 (reference: 13-EM-0399). Recruitment followed deferred consent processes, with written informed consent obtained by the study research team from parents/legal guardians within 24–48 hours20. The study reporting follows the Standards of Reporting of Diagnostic Accuracy Studies 2015 Update (https://www.equator-network.org/reporting-guidelines/stard/).  Procedures Data We used patient demographic data (sex at birth, age, self-reported ethnicity), clinical characteristics (reason for PICU admission, duration of acute illness prior to transport, pre-morbid Pediatric Overall Performance Category), details of infection status (including phenotyping for bacterial, viral, unknown and non-infectious illnesses using standardized published criteria21), clinical severity markers such as the Paediatric Index of Mortality (PIM-2)22 score, retrospectively applied Phoenix Sepsis Score (PSS)23 and paediatric acute respiratory distress syndrome (pARDS)24, at admission to PICU as appropriate, interventions performed (invasive ventilation, vasoactive drugs) during transport and PICU discharge outcome (vital status and duration of organ support) were extracted from the BASIC database.  Microarrays Whole blood was sampled into RNA-stabilising tubes (PAXgene, Qiagen, Germany) from recruited patients immediately during paediatric critical care retrieval. Samples were initially stored on ice packs (4ºC) prior to arrival to the admitting PICU, before storage at -80ºC and processing in batches using Illumina Human-HT-12 version 4 Expression BeadChips (Illumina, CA, USA).","Scaning - Arrays were scanned with an Illumina BeadArray Reader confocal scanner (BeadStation 500GXDW; Illumina, Inc., San Diego, CA) according to the manufacturer's instructions.","Labeling - Biotin-labeled cDNA samples for hybridization were prepared according to Illumina's recommended sample labeling procedure: 750 ng of total RNA was used for cDNA synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA using the Illumina TotalPrep RNA Amplification kit (Ambion, Inc., Austin, TX). cRNA concentrations were determined using a the Agilent BioAnalyzer system.","Nucleic Acid Extraction - 1. Objective This document describes the processes for extraction of total RNA, including miRNA, from PAXgene Tube 2. Scope This SOP applies to all staff who will be involved in PAXgene RNA extraction for the DIAMONDS study. 3. Hardware required  ITEM SUPPLIER CATALOGUE No. 3.1 PAXgene RNA Tube BD BD Ref: 762165 3.2 RNAse DNAse free cryovial (CRYO.S 4ml tube, external thread) Greiner Bio-one  Ref: 127278 3.3 QIAsymphony PAXgene blood miRNA extraction kit QIAgen 762635 3.4 50 ml conical-bottom tube Starlab E1450-0200 3.5 Multi-tube Vortex Various  3.6 Centrifuge Various  3.7 Microcentrifuge tubes Various  3.8 Heating block Various  3.6 10 ml serological pipette Starlab E4860-0010 3.7 Pipette tips (20 ul, 200 ul, 1000 ul) Starlab Various 3.8 10x10 cryoboxes (storage of 0.5ml RNA tubes) Sarstedt 95.64.997  Important points before starting • Maximum number of samples that can be run at a single time: 72 samples/day, 5h 45min machine time • Ideally this is a two-person job (one person preps the samples; the second person sets up the robot with consumables) Things to do before starting  1. Defrost PAXgene samples at room temperature, preferably overnight  (needed to be at room temperature for a minimum of 2 hours before extraction begins).  Safety note: Visually inspect each PAXgene tube before defrosting to check there are no cracks in the tube. The PAXgene tube should be placed in a 50ml falcon tube for defrosting and left behind the MSC. 2. Note down the volume of blood/cracks/Homemade PAXgene tubes prior to processing 3. Switch on the QIAsymphony, blue button on the lower left-hand side of the machine (initiation process takes approximately 15 minutes). 4. Prepare 2 vials of DNAase stock solution.  • Carefully add 900ul of RNase free water per vial and pipette up and down gently to mix (do not vortex) • Place the contents of the two vials (1.8ml in total) into the empty tube (labeled ETDN) in position 3 of the enzyme rack Note: DNase 1 solution can be stored at 2-8°C for up to 6 weeks 5. Vortex magnetic beads for 3 minutes and ensure that they are fully resuspended and place back in the reagent cartridge frame 6. Check the rest of the reagent trough and the BX2 bottle to ensure that they have not precipitated, if necessary warm to 37°C in a water bath to re-dissolve. 7. Mix buffer QSX2 with proteinase K depending on the number of samples • For 24 samples mix 7ml of QSX2 with 500µl of proteinase K • For 48, 14ml QSX2 with 1000µl of proteinase K • For 72, 21ml QSX2 with 1500µl of proteinase K  1. Setting up the QIAsymphony machine a. Login to the QIAsymphony, user: ‘Supervisor’, password: ‘diamonds5’ b. Start wizard. Select PAX -> RNA V5 and set number of samples to 48, press next Safety note: Do not open the front panel of the machine (clear plastic around touchscreen) during operation.  o Load waste drawer • Place 4 empty waste containers into slots Note: The empty rod cover and sample prep containers can be used as waste containers • Check that tip chute is loaded with the slope pointed towards the machine and that the sharps bin is sufficiently empty (The sharps bin for tips is in the cupboard under the machine on the right).  Note: It is ideal to seal and change the sharps bin regularly, as needed and always at the end of a 72-sample run. • Check that liquid waste container is empty. • Close drawer and complete inventory scan. o Load reagents drawer  • Label all new regent bottles/containers with the date (in case of a run of <24samples) • Load the reagents cartridge into the grey racks provided, if reagents cartridge is unopened attach “piercing lid”. If previously open, remove re- sealable lids carefully and place to the side. Safety note: Piercing lid is sharp, when handling lid only hold the sides, click onto reagent pack at both ends, do not press down (the machine will do this). Note: Ensure the re-sealable lid is numbered with corresponding trough number to prevent cross-contamination. Reagents cartridges can only remain open for 15 hrs. in total. Note down the length of time the cartridge is left open in the logbook placed on the QIAsymphony bench. • The lids for the three enzyme tubes can be placed under the enzyme tubes which slide into the side of the grey rack. • Slide the new grey rack containing the reagents cartridge into the consumable’s drawer. Note:  Partially used reagent cartridge can be stored until needed again. In such cases, based on the sample run (48/72 samples) slide the partially used cartridges in slot 1 at the front of the drawer, and the new reagent cartridge in slot 2  • Load rod covers and samples prep cartridges (refer to the table below) Note: If running 48 or more samples ensure that there are two full boxes of each. Number of samples 24 48 72 Reagent cartridges 1 1 2 Sample prep cartridges 27 54 81 8-Rod covers 3 6 9 1500ul tips 132 (5 racks) 264 (9 racks) 396 (13 racks) 200ul tips 28 (1 rack) 56 (2 racks) 84 (3 racks) • Load the pipette tips (refer table); place 4 of the blue 200µI racks into the slots to the back of the QIAsymphony and fill the rest of the slots with the 1500µl tips. Note: Do not discard the tip box. The empty tip container could be used for storage of unused tips after the run is complete. • Open bottle RDD and on the consumables page in the display screen, select “add bottle” and scan bottle barcode, then place the bottle in a consumable drawer (round hole). • Ensure that all consumables are correctly loaded and close drawer, when prompted, run inventory scan. • Load eluate drawer • Use 96 well PCR plate skirted/non skirted, (CAT NO.#AB0800/#AB0600) • On the display screen navigate to the eluate tab if not already there, select position 1, designate the collection format that will be used, and click on the box to enter plate info. •  Place 96 well plate in the cooling adaptor (PCR plate rack) slot of eluate drawer • Close drawer and complete inventory scan. 2. Sample preparation  a. Centrifuge samples for 10mins at  3500g for Commercial PAXgene tubes 4000g for homemade PAXgene tubes and commercial PAX with 1ml of blood Tip: When the first set of samples are spinning, prepare the buffer mix QSX2 with protinase K  3. Procedure 1. When samples have finished centrifuging process them in the MSC, discard the supernatant in liquid waste container (500ml Duran bottles), and blot dry the PAXgene tubes. 2. Resuspend the pellet in 300µl of the previously prepared, QSX2 - proteinase K mixture. Vortex for 30 seconds 3. Close the tubes with the Secondary Hemogard Closures provided and thoroughly resuspend the pellet by vertexing. For resuspension use a multitube vortex at full speed for 30s  4. Add 200µl of buffer BR2 to the resuspended pellet, close the tubes with the Secondary Hemogard closures and place them into the appropriate sample carrier  Safety note: Load tubes into the tube rack BEFORE removing the lids, support the rack with both hands and don’t raise above chest height when transferring it to the sample drawer. 5. Loading tube carrier into the QIAsymphony,  Note: It is easiest to load the tube carrier when stood in front of the QIAsymphony, with the tube rack on your left-hand side. • Open “sample drawer” the lanes should appear green • Slide tube carrier, up to the black line painted on the lanes, and wait for the green light to begin to flash. • Once green light has started to flash, slide the tube carrier fully into the lane. • The lane should flash orange, if the lane flashes red, check screen, and remove samples. Note: Repeatedly clicking OK on the display screen can result in samples getting stuck (always read error messages on the screen before clicking OK), if this occurs, restart machine. 5. Once the samples have been successfully loaded into the machine, use the screen to “select all’ and change samples information from the default type to PAXgene RNA, BD # 762165 in case of commercial PAXgene tube and SAR#62.551.201 for Homemade PAXgene tubes 6. Enter the sample ID for each sample by selecting a sample and click “change sample ID”, sample icon should now turn blue. Press NEXT. 7. Press select all and then select the assay protocol PAXgene > RNA V5. 8. Queue samples. 9. The run button should then appear, click RUN, the run should then start, per batch of 24 the run time just over 2:15 hours, for 48 it will be 4:30 hours. 10. Set up the program in PCR machine for heat inactivation of samples at 65°C 11. After the run is complete, login, open the elution drawer, remove elution plate, seal the wells, and place the elution plate in PCR machine at 65°C, for 10 minutes. Note:  If using Elution Microtubes CL (QIA#19588), heat an incubator to 80°C. If using 2 ml Sarstedt tubes, heat a shaker–incubator, heating block, or water bath to 65°C. Do not exceed the incubation time or temperature. 12. After incubation, chill immediately on ice and if the RNA eluate will not be used immediately, store at -80°C.  Tip: As samples will need to be quantified it is advisable at this time to aliquot 20ul of each sample into 0.5ml Eppendorf tubes. 13. In the meantime, on the QIAsymphony, navigate to the elution page, and click the ‘remove elution plate’ option, and let the QIAsymphony scan the drawer. 14. Insert memory stick, (near ON switch) navigate to > Tools >results > import/export for the aliquot positions. 4. After the QIAsymphony run • Remove all the consumables, e.g. Sample tubes, reagents cartridges, consumables, waste containers, tip racks, pipette waste chute and tip stand, select “scan later” when prompted for each drawer. • Place the re-sealable seals on the reagent cartridges, these can be stored at room temperature (in case of a run with less than 48 samples the cartridge could be re-used)  • Seal back the enzymes with respective lids, and these should be stored in the fridge at 4°C. • Remove all PAXgene tubes from the sample carrier and discard them in sharps bin. • Discard the used sample prep cartridges and 8-rod covers by sealing the waste containers with lids • Spray 70% ethanol onto paper towel and carefully wipe the entire machine deck. • Remove liquid waste container, pour waste into Duran bottle and add 10% Virkon. Leave disinfectant solution to stand for 30 min before disposing in laboratory sink.  • Disinfect the liquid waste container by spraying distel disinfectant and thoroughly wiping them with tissue paper before loading into the machine. • Remove both parts of the tip chute and spray with distel disinfectant, leave to stand on tissue paper for 30 min. • Perform a 30-minute UV scan; Tools > maintenance > UV scan (plastic cover of the symphony has a UV filter built in). • Replace both parts of the tip chute • Check sharps bin under tip chute. Seal and replace if necessary. • Ensure that all the QIAsymphony drawers are shut and switch off the machine. • Autoclave the duran bottle containing the liquid waste from PAXgene tubes (step 1) according to standard operating procedure."],"figure_sub":["MIAME Score","Raw Data","Organization","Assays and Data","Processed Data","MAGE-TAB Files","Array Designs"],"data_protocol":["Data Transformation - Please see the accompanying Rmd file attached to this submission."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"study_type":["transcription profiling by array"],"species":["Homo sapiens"],"pubmed_title":["Host gene expression prior to emergency paediatric intensive care admission predicts severity and shared immune dysfunction in an inclusive population of critically ill children"],"pubmed_authors":["Michael J. Carter, Joshua Hageman, Yael Feinstein, Jethro Herberg, Dominic Habgood-Coote, Victoria Wright, Samuel Nichols, Nazima Pathan, Naomi Edmonds, Katie Burnham, Alexander Mentzer, Julian Knight, Michael Levin, Myrsini Kaforou, Simon Nadel, Mark J. Peters, Padmanabhan Ramnarayan","Michael Carter"],"additional_accession":[]},"is_claimable":false,"name":"Host gene expression profiling on children with any cause critical illness at retrieval or admission to paediatric critical care","description":"We analysed data collected as part of the Biomarkers of Acute Serious Illness in Children (BASIC) study. BASIC was a prospective cohort study that enrolled critically ill children admitted to four PICUs in London and Eastern England, UK, during emergency transport by the Children’s Acute Transport Service (CATS), the regional paediatric critical care retrieval service from 2014–2016. We aimed to identify, and validate, shared features of immune dysfunction (\\\"endotypes\\\") and assess association with clinically important outcomes (primary outcome: ventilator-free days at day 30). Whole blood was sampled into RNA-stabilising tubes (PAXgene, Qiagen, Germany) from recruited patients immediately during paediatric critical care retrieval. Samples were initially stored on ice packs (4ºC) prior to arrival to the admitting PICU, before storage at -80ºC and processing in batches using Illumina Human-HT-12 version 4 Expression BeadChips (Illumina, CA, USA). Statistical analysis was undertaken in R: A Language and Environment for Statistical Computing (version 4.4.1). Microarray data were compiled, log2 transformed and normalised. Of 384 samples assay, 382 met quality control criteria and went forwards for bioinformatics analysis. We used the top 15% (~5000) most variable transcript probes in the dataset before using k-means clustering (Hartigan and Wong algorithm) with two centres to determine BASIC endotype membership for individual children at retrieval to PICU. BASIC endotype membership was internally validated with a train/validation (2/3 training, 1/3 validation) partition of the dataset, and a nested cross-validation implementation of GLMNet. We identified two robust endotypes, BASIC endotype 1 (122, 31.9%, children), and BASIC endotype 2 (260, 68.1%, children), present in children with diverse illnesses and age groups. BASIC endotype 1 membership was associated with 4.1 (95% CI 2.0–6.2) days of increased length of mechanical ventilation and a non-significant association with mortality. BASIC endotype 1 membership was associated with increase naïve and resting memory CD4 T cell proportions, lower neutrophil proportions and lower gene expression sets associated with tumour necrosis factor (TNF)-α, interferon-γ, interferon-α, and interleukin-6/JAK/STAT pathways in comparison with BASIC endotype 2. These BASIC endotypes may enable stratified trials of treatment for immune dysfunction.","dates":{"release":"2025-09-30T00:00:00Z","modification":"2025-09-30T01:04:50.863Z","creation":"2025-05-27T08:27:02.479Z"},"accession":"E-MTAB-15164","cross_references":{"EFO":["EFO_0002768","EFO_0002944","EFO_0003814","EFO_0003813","EFO_0005518","EFO_0003816","EFO_0003815"]}}