- Basic aspects of thoracic anaesthesia are general anesthesia often combined with regional anesthesia, intubation with double lumen tube and separation of lung ventilation. Proper assessment of pain and adequate analgesia in intraoperative and postoperative period is a challenging issue for medical practitioners.
- Intraoperative trauma may lead to many metabolic implications and disturbance of haemostasis, what can be reflected in change of blood and saliva hormone and other substance levels, such as alpha-amylase, cortisol, testosterone, secretory IgA, β-endorphin, nerve growth factor, calcitonin gene-related protein and P substance.
- The aim of this study is to assess the impact of regional anesthesia on hormone levels in postoperative period. Saliva was collected from participants in order to perform laboratory tests, using a special disposable Salivette tube (Sarstedt AG & Co, Germany). Saliva was collected by placing a sterile tampon under the tongue or chewing for 30-45 seconds.
- The soaked saliva pad was then placed in a suspended insert with a perforated bottom. The insert with a tampon was placed in a centrifuge tube and closed with a stopper. Next the tube was centrifuged (1000 x g for 10 min.) to obtain a ready to test saliva supernatant. Approximately 0,7 ml of the supernatant from every sample collected was used for further testing. Samples were frozen after centrifugation at – 85°C until performing laboratory tests. Blood was collected for laboratory tests from the ulnar vein. Blood for testing was collected using disposable equipment in a volume of 5ml into a tube containing ethylenediaminetetraacetic acid (EDTA) and aprotinin.
- Next the tube was centrifuged (1000 x g for 5 min.). After centrifugation and separation of Centrifuge tube sizes morphotic elements, the obtained plasma was divided into two tubes and frozen at – 85°C until performing laboratory tests.
Immune Responsiveness and Outcome After Aortic Valve Surgery (Measure)
- Barts Heart Centre is a 255-bedded specialist cardiac centre where approximately 200 primary isolated open aortic valve replacement procedures are performed per year. It is closely associated both geographically and academically with the William Harvey Research Institute (WHRI), which forms part of Queen Mary University, London (QMUL). This will allow laboratory analysis of all collected blood samples within 15 minutes if necessary. Barts Heart Centre is one of the largest cardiac units in Europe and a key research objective of QMUL/WHRI is to support cardiac research.
Sample collection and storage:
Following written informed consent all patients will be pre-operatively risk-scored by means of ASA, Euroscore 2 and conventionally risk-scored for the development of surgical site infection.
- Blood (44ml total) will be taken from 150 aortic valve replacement patients immediately prior to surgery (T0).
- Blood (7ml) will be collected into a PAXgene tube and stored at -80° for later genetic analysis. A clotted sample (7ml) will be collected in a standard gold top SST tube, left to clot then centrifuged for 10 mins at 1000g, the serum separated and stored at -80° for later analysis of antibody levels.
- In addition, 30 ml blood will be taken for study of peripheral blood mononuclear cells (PBMCs), drawn into standard purple top EDTA tubes (anticoagulant). These will be separated within 3 hours of collection by Ficoll-Paque density gradient centrifugation, whereby PBS-diluted blood is carefully layered over Ficoll and spun brake off in a centrifuge at 400g for 30 minutes.
- The PBMC layer is then extracted and washed twice in PBS and stored in 10% dimethyl sulfoxide freezing solution in liquid nitrogen so that they can be thawed, and batch analysed at a later date.
Sample analysis:
PBMCs will be counted using a haemocytometer, then cultured in RPMI and 10% serum with either LPS, α-toxin or unstimulated for 24 hours, after which they will be analysed immediately as detailed below.
• Soluble Mediators (Plasma)
– Storage: Ethylenediaminetetraacetic acid (EDTA, BD Biosciences Vacutainer, 9mL) anticoagulated blood will be centrifuged (1200g, 10mins, 20°C) and plasma stored at -80°C within 2hrs of collection
– Analysis: Cytokines: Meso Scale Discovery (MSD) V-PLEX Proinflammatory Panel 1 will be employed to quantify IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, and TNF-α. Plates will be read using a MSD QuickPlex SQ 120 imager (Wiliam Harvey Institute, QMUL).
Antibodies: These will be measured by means of ELISA assays against staphylococcal antigens (α-toxin, teichoic acid) and core moieties of the endotoxin molecule (EndoCAb).
– Whole blood LPS/α-toxin Stimulated Cytokine Release
– Technique: As previously described and validated in our laboratory, heparinized blood will be stimulated for 4hrs (37°C, 250rpm) with 1ng/ml LPS within 2hrs of draw. After incubation, samples will be centrifuged (1200g, 10mins, 20°C) and supernatant stored at -80°C.
– Cytokine quantification: As per ‘soluble mediators’ MSD V-PLEX Proinflammatory Panel 1 will be employed to quantify TNF-α (primary outcome as metric of immune competence) and other cytokines. Cytokines will be expressed as a factor of the number of circulating monocytes.
Flow Cytometric Immunophenotyping and Functional Assessment of Leukocytes
Flow Cytometry: All samples will be analysed on the same machine (Becton Dickinson (BD) Fortessa, Rayne Building, UCL). Standardisation and comparison of output will be achieved via employment of constant voltages and compensation matrix throughout with daily matching of values to a single batch of Cytometry Setup and Tracking (CS&T) beads (BD). Leukocyte cell surface staining will be conducted using antibodies principally supplied by BD. Staining, data capture and storage will be conducted in accordance with a single study standard operating procedure. 5 panels (up to 14-colour) and two functional assays will be performed at each time-point. Data will be analysed in FlowJo version 10.5.
Surface Marker Staining:
Quantification and calibration panel: CD45, CD56, CD3, CD4, CD8, CD19, CD14, CD16, performed in a BD TruCount tube to enable enumeration of cell subsets Cell specific panels (incorporating markers of sub-categorisation and markers of immune competence)
– Monocyte/Myeloid: CD3, CD19, CD56, CD163, CD16, CD33, CD141, CD206, CD274, CD14, CD1c, CD11c, HLA-DR, CD80, CD123, CD83
– Neutrophil: CD3, CD19, CD56, HLA-DR, CD64, CD62L, CD11b, CD88, CD66b, CD14, CD11c, CD16, CD184, CD182
– Lymphocyte: CD19, CCR6, CD45RA, CD3, CD4, CD8, CXCR3, CD127, CD62L, CD25, CD56, CD27, CD279 HLA-DR Expression: Will be quantified on CD14+ monocytes using BD QuantiBrite beads.
Functional Measures Phagocytosis: The ability of neutrophils and monocytes to phagocytose opsonised FITC-labelled E.coli bacteria will be quantified via the PhagoTest assay (BD) as per manufacturer’s instructions Reactive oxygen ‘burst’: Quantitative determination of leukocyte oxidative burst will be undertaken using the PhagoBurst assay (BD) in response to un-labelled opsonized E.coli, phorbol 12-myristate 13-acetate (PMA) and the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP).
The pre-surgery sample PBMCs that will be stimulated with LPS (type and concentration to be optimized via dose response curve on healthy volunteers) and α-toxin will undergo flow cytometry to measure TLR4 and TLR5 surface expression, HLA-DR surface expression and quantification of NF-kB using appropriate antibodies validated in the literature.
Centrifuge Tubes, 50 ml ,PP,Sterile,Plug-Seal Cap, in Bag |
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| LC8005-500 | GenDepot | 500/CS |
Centrifuge Tubes, 15 ml ,PP,Sterile,Plug-Seal Cap, in Bag |
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| LC8003-500 | GenDepot | 500/CS |
Centrifuge Tubes, 50 ml , Black ,PP,Sterile |
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| LC8008-025 | GenDepot | 25/Bag |
Centrifuge Tubes, 50 ml , Black ,PP,Sterile |
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| LC8008-500 | GenDepot | 500/CS |
Centrifuge Tubes, 15 ml , Black ,PP,Sterile |
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| LC8007-050 | GenDepot | 50/Bag |
Centrifuge Tubes, 15 ml , Black ,PP,Sterile |
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| LC8007-500 | GenDepot | 500/CS |
Centrifuge Tubes, 50 ml ,PP,Sterile,Plug-Seal Cap,Self Standing, in Bag |
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Corning LSE Single Block 5 x 50 mL Centrifuge Tubes |
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16 mm Test Tube Foam Rack (for 15 mL centrifuge tubes) |
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Centrifuge Tubes, 50 ml ,PP,Sterile,Plug-Seal Cap, in Rack |
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| LC8004-500 | GenDepot | 500/CS |
Centrifuge Tubes, 15 ml ,PP,Sterile,Plug-Seal Cap, in Rack |
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| LC8002-500 | GenDepot | 500/CS |
Block, 15 x1.5ml centrifuge tubes |
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| BCM1405-B15 | Bio Basic | 1 pcs, 1 UNIT |
Block, 15 x1.5ml centrifuge tubes |
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50ml Conical Centrifuge Tubes, Green Cap, 25/Bag |
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| CT788-G | Bio Basic | 1PK, 25UNIT |
50ml Conical Centrifuge Tubes, Yellow Cap, 25/Bag |
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| CT788-Y | Bio Basic | 1PK, 25UNIT |
Eppendorf Adapter for 0.5 mL micro- centrifuge tubes + 0.6 ml Microtainer set of 6 |
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4-Ways Microtube and Centrifuge Tubes Racks, Natural |
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| BSW10 | Benchmark Scientific | 1 PC |
Block, 24 x 0.5ml centrifuge tubes |
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| BCM1405-B05 | Bio Basic | 1 pcs, 1 UNIT |
Block, 24 x 0.5ml centrifuge tubes |
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| BCM1412-BSW05 | Bio Basic | 1 pcs, 1 UNIT |
Block, 24 x 0.5ml centrifuge tubes |
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Block, 24 x 0.5ml centrifuge tubes |
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Falcon Conical 50ml PP Centrifuge Tubes Bagged |
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Block, 12 x 15ml centrifuge tubes |
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| BSW15 | Benchmark Scientific | 1 PC |
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Block, 24 x1.5ml centrifuge tubes (conical) |
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| CEN2051 | Scientific Laboratory Supplies | PK200 |
Benchmark Centrifuge Tube Yellow 5mL (4 bags of 50) |
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| CEN2049 | Scientific Laboratory Supplies | PK200 |
At the end of stimulation, cells will be harvested, and RNA extracted for quantitative (q)PCR to evaluate AID mRNA expression. Although B cells in the PBMC cultures have been stimulated in the presence of other cell types, primarily T cells and monocytes-macrophages, our endpoint is to measure a B-cell response, as AID is exclusively expressed in B cells.