Immune Responsiveness and Outcome After Aortic Valve Surgery.

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 Centrifuge tube 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.

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.

The Impact of Regional Anaesthesia on Hormone Levels in Thoracic Surgery.

• 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.

Centrifuge Tubes, 50 ml , Black ,PP,Sterile
LC8008-025	GenDepot	25/Bag
Centrifuge Tubes, 50 ml , Black ,PP,Sterile
LC8008-500	GenDepot	500/CS
Centrifuge Tubes, 15 ml , Black ,PP,Sterile
LC8007-050	GenDepot	50/Bag
Centrifuge Tubes, 15 ml , Black ,PP,Sterile
LC8007-500	GenDepot	500/CS
Centrifuge Tubes, 50 ml ,PP,Sterile,Plug-Seal Cap, in Bag
LC8005-500	GenDepot	500/CS
Centrifuge Tubes, 15 ml ,PP,Sterile,Plug-Seal Cap, in Bag
LC8003-500	GenDepot	500/CS
Centrifuge Tubes, 50 ml ,PP,Sterile,Plug-Seal Cap,Self Standing, in Bag
LC8006-500	GenDepot	500/CS
Test Tube rack for 15 x 50 ml tubes
 -B2000-4-T500	Benchmark Scientific	1 PC
Centrifuge Tubes, 50 ml ,PP,Sterile,Plug-Seal Cap, in Rack
LC8004-500	GenDepot	500/CS
Centrifuge Tubes, 15 ml ,PP,Sterile,Plug-Seal Cap, in Rack
LC8002-500	GenDepot	500/CS
COMBI TUBE HOLDER, 6X15/50 ML TUBES
480148	CORNING	1/pk
ERLENMEYER FLASK, 50 ML, PP
4985P-50	CORNING	6/pk
ERLENMEYER FLASK, 50 ML, PMP
4990P-50	CORNING	6/pk
Block, 15 x1.5ml centrifuge tubes
BCM1405-B15	Bio Basic	1 pcs, 1 UNIT
500 ML DMEM (DULBECCO'S MODIFICATION OF EAGLE'S MEDIUM)/F12 50:50 MIX WITHOUT L-GLUTAMINE
15-090-CV	CORNING	500 mL/pk
0.2 ml 8-Strip Tubes, 120 Strips of 8 Tubes
GP301	GenDepot	960 tubes
0.2 ml 8-Strip Tubes, 250 Strips of 8 Tubes
GP302	GenDepot	2,000 tubes
0.2 ml 12-Strip Tubes, 80 Strips of 12 Tubes
GP305	GenDepot	960 tubes
Naked Gold 40 nm Concentrated Gold Sol, 50 O.D./mL, 1 mL
NGL40-B001	BioAssayWorks	1 mL
Naked Gold 40 nm Concentrated Gold Sol, 50 O.D./mL, 9 mL
NGL40-B009	BioAssayWorks	9 mL
Naked Gold 40 nm Concentrated Gold Sol, 50 O.D./mL, 44 mL
NGL40-B044	BioAssayWorks	44 mL
Naked Gold 40 nm Concentrated Gold Sol, 50 O.D./mL, 100 mL
NGL40-B100	BioAssayWorks	100 mL
Test Tube rack for 30 x 50 ml tubes
  B2000-8-T500	Benchmark Scientific	1 PC
Test Tube rack for 40 x 0.5 ml tubes
B2000-4-T5	Benchmark Scientific	1 PC
FALCON® 15 ML POLYSTYRENE CENTRIFUGE TUBE, CONICAL BOTTOM, WITH DOME-SEAL SCREW CAP, STERILE, 50/BAG, 500/CASE
352095	CORNING	50/pk
0.2 ml 8-Strip Tubes & Flat Caps, 250 Strips of 8 Tubes and Caps
GP203	GenDepot	2000 tubes/caps
0.2 ml 8-Strip Tubes & Domed Caps, 120 Strips of 8 Tubes and Caps
GP204	GenDepot	960 tubes/caps
0.2 ml 8-Strip Tubes & Domed Caps, 250 Strips of 8 Tubes and Caps
GP205	GenDepot	2,000 tubes/caps
0.3 ml 8-Strip Tubes & Domed Caps, 250 Strips of 8 Tubes and Caps
GP209	GenDepot	2,000 tubes/caps
0.2 ml 12-Strip Tubes & Flat Caps, 80 Strips of 12 Tubes and Caps
GP210	GenDepot	960 tubes/caps
0.2 ml 12-Strip Tubes & Domed Caps, 80 Strips of 12 Tubes and Caps
GP211	GenDepot	960 tubes/caps
0.2 ml 8-Strip Tubes & Flat Caps, 125 Strips of 8 Tubes and Caps
GP903	GenDepot	1000 tubes/caps
0.2 ml 8-Strip Tubes & Dome Caps, 125 Strips of 8 Tubes and Caps
GP904	GenDepot	1000 tubes/caps
Naked Gold 40 nm Concentrated Gold Sol, 15 O.D./mL, 1 mL
NG40-B001	BioAssayWorks	1 mL
Naked Gold 40 nm Concentrated Gold Sol, 15 O.D./mL, 9 mL
NG40-B009	BioAssayWorks	9 mL
Naked Gold 40 nm Concentrated Gold Sol, 15 O.D./mL, 44 mL
NG40-B044	BioAssayWorks	44 mL
Naked Gold 40 nm Concentrated Gold Sol, 15 O.D./mL, 100 mL
NG40-B100	BioAssayWorks	100 mL
MINI BIOREACTOR, 50 ML CENTRIFUGE TUBE WITH VENT CAP, STERILE
431720	CORNING	25/pk
Test Tube rack for 41 x 15 ml tubes
  B2000-4-T150	Benchmark Scientific	1 PC
Test Tube rack for 76 x 15 ml tubes
  B2000-8-T150	Benchmark Scientific	1 PC
50 ml Reagent Reservoir
GR238016	Genorise Scientific	Pack of 200
50ml Conical Centrifuge Tubes, Green Cap, 25/Bag
CT788-G	Bio Basic	1PK, 25UNIT
50ml Conical Centrifuge Tubes, Yellow Cap, 25/Bag
CT788-Y	Bio Basic	1PK, 25UNIT
0.2 ml 8-Strip Tubes & Optically Clear Caps, 120 Strips of 8 Tubes and Caps
GP207	GenDepot	960 tubes/caps
Test Tube rack for 40 x 1.5/2.0 ml tubes
  B2000-4-T1520	Benchmark Scientific	1 PC
ML 228
A4508-50	ApexBio	50 mg
ML-204
9448-50	Biovision
ML 213
B5628-50	ApexBio	50 mg
ML 171
B5672-50	ApexBio	50 mg
ML 204
B5696-50	ApexBio	50 mg
ML SA1
B5700-50	ApexBio	50 mg
ML 190
B5741-50	ApexBio	50 mg
ML 337
B5786-50	ApexBio	50 mg
ML 145
B7604-50	ApexBio	50 mg
ML 154
B7765-50	ApexBio	50 mg
ML 348
B4933-50	ApexBio	50 mg
ML-090
C4746-50	ApexBio	50 mg
ML-335
C4792-50	ApexBio	50 mg
ML-297
C5045-50	ApexBio	50 mg
0.2 ml 8-Strip Low Profile Tubes, 120 Strips of 8 Tubes
GP303	GenDepot	960 tubes
0.2 ml 12-Strip Low Profile Tubes, 80 Strips of 12 Tubes
GP304	GenDepot	960 tubes
0.2 ml 8-Strip Low-Profile Tubes & Flat Caps, 120 Strips of 8 Tubes and Caps
GP201	GenDepot	960 tubes/caps
0.2 ml 8-Strip Low-Profile Tubes & Domed Caps, 120 Strips of 8 Tubes and Caps
GP202	GenDepot	960 tubes/caps
0.2 ml 8-Strip Tubes & Domed Caps, Assorted Colors, 250 Strips of 8 Tubes and Caps
GP206	GenDepot	2,000 tubes/caps
0.2 ml 8-Strip White Tubes & Optically Clear Caps, 120 Strips of 8 Tubes and Caps
GP208	GenDepot	960 tubes/caps
AXYPREP MAG DYECLEAN KIT- 50 ML - 5000 PREPS
MAG-DYECL-50	CORNING	1/pk
RESERVOIR, POLYPROPYLENE, 50 ML, V-BOTTOM, NONSTERILE, BULK
RES-V-50	CORNING	100/pk
50 ML AMPHOTERICIN B, LIQUID 250 µG/ML SOLUBILIZED
30-003-CF	CORNING	50 mL/pk
10 ML GENTAMICIN SULFATE, LIQUID 50 MG/ML SOLUTION
30-005-CR	CORNING	10 mL/pk
50 ML KANAMYCIN SULFATE, LIQUID, 5,000 µG/ML SOLUTION
30-006-CF	CORNING	50 mL/pk
Dressed Gold Streptavidin at 50 O.D./mL, 1 mL
DSTAL-B001	BioAssayWorks	1 mL
Dressed Gold Streptavidin at 50 O.D./mL, 9 mL
DSTAL-B009	BioAssayWorks	9 mL
Dressed Gold NeutrAvidin at 50 O.D./mL, 1 mL
DNTAL-B001	BioAssayWorks	1 mL
Dressed Gold NeutrAvidin at 50 O.D./mL, 9 mL
DNTAL-B009	BioAssayWorks	9 mL
Antigen Retrieval Solution, Citrate pH 6.0 (50 ml, 100X)
ARS6-50	Alpha Diagnostics	5 L
Antigen Retrieval Solution, EDTA pH 8.0 (50 ml, 100X)
ARS8-50	Alpha Diagnostics	5 L
Chloramphenicol Solution (50 mg/ml)
C090-10ML	TOKU-E	10 mL
Chloramphenicol Solution (50 mg/ml)
C090-10x10ML	TOKU-E	10 x 10 mL
BrightGreen miRNA qPCR MasterMix-Low ROX
MasterMix-mL	ABM	4 x 1.25 ml for 500 reactions
Rat Monoclonal Anti-Mouse CD4 Pur. Low Endotoxin (Clone YTS 191.1) (rat IgG2b)
MCD004-ML	Alpha Diagnostics	100 ug
Rat Monoclonal Anti-Mouse CD25 , Pur. Low Endotoxin (Clone PC61.5.3) (rat IgG1)
MCD025-ML	Alpha Diagnostics	100 ug
ML-7 hydrochloride
A3626-50	ApexBio	50 mg
ML 218 hydrochloride
B5622-50	ApexBio	50 mg
ML 120B dihydrochloride
B5756-50	ApexBio	50 mg
ML 9 hydrochloride
B6300-50	ApexBio	50 mg
ML 10302 hydrochloride
B7413-50	ApexBio	50 mg
ML 00253764 hydrochloride
B7715-50	ApexBio	50 mg
AXYPREP FRAGMENTSELECT-I KIT- 50 ML - 380 PREPS
MAG-FRAG-I-50	CORNING	1/pk
RESERVOIR, POLYPROPYLENE, 50 ML, V-BOTTOM, STERILE, BULK
RES-V-50-S	CORNING	5/pk
Dressed Gold Streptavidin at 15 O.D./mL, 1 mL
DSTA-B001	BioAssayWorks	1 mL
Dressed Gold Streptavidin at 15 O.D./mL, 9 mL
DSTA-B009	BioAssayWorks	9 mL
Dressed Gold NeutrAvidin at 15 O.D./mL, 1 mL
DNTA-B001	BioAssayWorks	1 mL
Dressed Gold NeutrAvidin at 15 O.D./mL, 9 mL
DNTA-B009	BioAssayWorks	9 mL
Antigen Retrieval Solution, Tris-EDTA pH 9.0 (50 ml, 100X)
ARS9-50	Alpha Diagnostics	5 L
Dressed Gold Protein A at 50 O.D./mL, 1 mL
DPRAL-B001	BioAssayWorks	1 mL
Dressed Gold Protein A at 50 O.D./mL, 9 mL
DPRAL-B009	BioAssayWorks	9 mL
Dressed Gold Protein G at 50 O.D./mL, 1 mL
DPRGL-B001	BioAssayWorks	1 mL
Dressed Gold Protein G at 50 O.D./mL, 9 mL
DPRGL-B009	BioAssayWorks	9 mL
Dressed Gold Protein L at 50 O.D./mL, 1 mL
DPRLL-B001	BioAssayWorks	1 mL
Dressed Gold Protein L at 50 O.D./mL, 9 mL
DPRLL-B009	BioAssayWorks	9 mL

Next the tube was centrifuged (1000 x g for 5 min.). After centrifugation and separation of  morphotic elements, the obtained plasma was divided into two tubes and frozen at – 85°C until performing laboratory tests.