SAMPLE TAKING AND STORAGE
* BAL:
* Technique: The bronchoscope will be inserted in segment 6, aliquots of sterile saline will be instilled. The procedure will be repeated in segments 3 and 5, in the case of the right lung, and in segment 6 and lingula in the left lung. 20-30cc of BAL will be completed for each graft.
* Storage: Samples will be frozen immediately after collection and transferred to the laboratory with a cold accumulator to ensure that they do not thaw.
* Analysis: The lung microbiome will be analysed.
* Lung biopsy:
* Technique: Lung biopsy of a minimum of 2cm3 will be taken using non-absorbable automatic suture.
* Storage: Samples will be preserved in RNA Stabilization solution Reagent (QIAGEN), which allows specimens to be kept at room temperature until frozen, avoiding RNA degradation.
* Analysis: RNA sequencing of transcription of inflammatory signals.
* Ex vivo perfusion liquid solution:
* Technique: Taking samples sterile with a 20cc syringe.
* Storage: Samples will be frozen immediately after collection and transferred to the laboratory with a cold accumulator to ensure that they do not thaw.
* Analysis: Microbiome analysis
* Preservation liquid solution:
* Technique: Taking samples sterile with a 20cc syringe.
* Storage: Samples will be frozen immediately after collection and transferred to the laboratory with a cold accumulator to ensure that they do not thaw.
* Analysis: Microbiome and cytokine analysis
All samples will be stored in a freezer at -80
\- Microbiome analysis For the analysis of microbial diversity, the V4 variable region of the 16S gene will be amplified from bacterial DNA by PCR. Amplicons will be sequenced using Illumina (MiSeq) technology. Samples will be processed and more than 10,000 300bp sequences per sequence per sample will be generated.
Gene expression analysis RNA extraction will be performed using the commercial RNeasy Mini Kit (QIAGEN). Gene expression analysis will be performed by quantitative PCR (qPCR) using the predesigned TransplantRejection panel from SignArray (AnyGenes®, Paris, France) that includes 84 genes that have been described to be related to the immune response in transplant rejection. These are: Genes included in the panel: CX3CR1, ICAM1, ITGA2, ITGAE, ITGAM, PECAM1, THBS1, THBS2, VCAM1, COL1A2, CCR5, CCR7, CD40, CD40LG, CD80, CD86, CTLA4, CXCR3, STAT4, TGFB1, CD44 , CTGF, MMP1, MMP2, MMP7, MMP9, BMP7, CCL11, CCL2, CCL3, CCL4, CCL5, CSF2, CXCL10, IFNG, IL10, IL12A, IL13, IL16, IL1B, IL2, IL2RA, IL3, IL32, IL4, IL5 , IL6, IL8, TNF, TGFB2, TGFB3, TIMP1, VEGFA, MS4A1, CXCL11, CXCL9, CXCR4, ADAM17, C3, CASP1, CASP3, CASP8, CCR2, CCR3, CD14, CD28, CD8A, FAS, FASLG, FCGR1A, GZMA , GZMB, NFKB1, NOS2, PRF1, PSMB9, STAT1, STAT6, TAP1, TLR3, TLR4, TLR9, TNFAIP3, TNFSF10.
\- Cytokine analysis The determination of cytokines in the perfusion fluid will be carried out using immunoassays based on Luminex™ xMAP™ technology (multi-analyte profiling) that allow the simultaneous quantification and detection of different secreted proteins (cytokines, chemokines, growth factors, etc.) We will use panels designed specifically for the gene products of interest.
Cytokine levels are measured using an immunoassay based on Luminex™ xMAP™ technology that allows for multi parametric analysis of the different cytokines. To this end, a personalized cytokine panel is designed based on the published literature on the effect of statins on the production of cytokines and other proinflammatory chemokines at a systemic level, as well as bibliographic evidence on the cytokines involved in lung transplantation. The cytokines analysed in the panel designed for this purpose are IFN gamma, IL-1 beta, IL-6, IL-8 (CXCL8), IL-18, IP-10 (CXCL10), MCP-1 (CCL2), MIP- 1 alpha (CCL3), TNF alpha, VEGF-D (Custom Procartaplex Multiplex Panel, Invitrogen, ThermoFisher Scientific, MA, USA). The immunological analysis will be carried out using the Invitrogen ProcartaPlex Analyst 1.0 software, supplied with the reagents.
\- Bioinformatic analysis of the sequences To obtain the microbial composition of each sample, we will use the QIIME software. QIIME is a software pipeline that uses phylogenetic information and multivariate statistical techniques to compare microbial communities and determine, for example, whether they are statistically different. The program also identifies the species that contribute the most to these differences and discovers patterns of various types that characterize specific groups of samples.
