Ignite Creation Date:
2024-05-06 @ 2:50 PM
Last Modification Date:
2024-10-26 @ 1:37 PM
Study NCT ID:
NCT04430972
Status:
NOT_YET_RECRUITING
Last Update Posted:
2023-01-26
First Post:
2020-05-20
Brief Title:
Immune Responsiveness and Outcome After Aortic Valve Surgery Measure
Sponsor:
Barts The London NHS Trust
Organization:
Barts The London NHS Trust
Study Overview
Official Title:
Is Pre-operative Impaired imMune rEsponsiveness Associated With Adverse Outcome Following Aortic Valve Replacement SURgEry MEASURE
Status:
NOT_YET_RECRUITING
Status Verified Date:
2022-04
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
Measure
Brief Summary:
There is considerable morbidity and mortality associated with cardiac surgery Currently little effort is made to quantify how well the immune system of an individual can cope with inflammation or infection to which they are exposed during surgery
The investigators have previously demonstrated that having higher pre-operative antibody levels is associated with a lower risk of infection and a shorter stay in hospital after cardiac surgery
The investigators aim to study 150 patients undergoing aortic valve replacement and explore their dynamic immune responsiveness The investigators will determine if this response is correlated with the post-operative outcome development of post-operative infection or increased length of hospital stay
The investigators will compare this response with the previously measured static markers of immune competence and also with a novel device that may give a more rapid measure of dynamic immunity
The investigators will approach patients in the cardiac surgical pre-assessment clinic to see if they are willing to participate in the study
Immediately once under anaesthetic blood will be taken for testing and then again at the end of surgery 24h after surgery at discharge from hospital and at follow-up clinic approximately 4 weeks later There will be no additional needle insertions on top of those routinely performed The investigators will collect data from the routine observations as far as 1 year after surgery
If the investigators can show an association between immune function and subsequent post-operative outcome it may be possible to determine ways to improve outcomes for patients undergoing heart surgery
This might include better information on risks and benefits of surgery actively boosting immune function vaccination immune-nutrition passively improving immunity administering antibodies or consider current alternatives to open heart surgery where the threat of infection or inflammation may be markedly reduced eg trans-catheter aortic valve implantation
The investigators have previously demonstrated that having higher pre-operative antibody levels is associated with a lower risk of infection and a shorter stay in hospital after cardiac surgery
The investigators aim to study 150 patients undergoing aortic valve replacement and explore their dynamic immune responsiveness The investigators will determine if this response is correlated with the post-operative outcome development of post-operative infection or increased length of hospital stay
The investigators will compare this response with the previously measured static markers of immune competence and also with a novel device that may give a more rapid measure of dynamic immunity
The investigators will approach patients in the cardiac surgical pre-assessment clinic to see if they are willing to participate in the study
Immediately once under anaesthetic blood will be taken for testing and then again at the end of surgery 24h after surgery at discharge from hospital and at follow-up clinic approximately 4 weeks later There will be no additional needle insertions on top of those routinely performed The investigators will collect data from the routine observations as far as 1 year after surgery
If the investigators can show an association between immune function and subsequent post-operative outcome it may be possible to determine ways to improve outcomes for patients undergoing heart surgery
This might include better information on risks and benefits of surgery actively boosting immune function vaccination immune-nutrition passively improving immunity administering antibodies or consider current alternatives to open heart surgery where the threat of infection or inflammation may be markedly reduced eg trans-catheter aortic valve implantation
Detailed Description:
Experimental design and methods
Setting
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 QMULWHRI 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 20C and plasma stored at -80C 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 LPSα-toxin Stimulated Cytokine Release
Technique As previously described and validated in our laboratory heparinized blood will be stimulated for 4hrs 37C 250rpm with 1ngml LPS within 2hrs of draw After incubation samples will be centrifuged 1200g 10mins 20C and supernatant stored at -80C
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 CST 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 105
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
MonocyteMyeloid 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 Ecoli bacteria will be quantified via the PhagoTest assay BD as per manufacturers instructions Reactive oxygen burst Quantitative determination of leukocyte oxidative burst will be undertaken using the PhagoBurst assay BD in response to un-labelled opsonized Ecoli phorbol 12-myristate 13-acetate PMA and the chemotactic peptide N-formyl-Met-Leu-Phe fMLP
o 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 qPCR 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
Enough healthy volunteer blood is available so that all above techniques can be adequately practiced and optimized prior to patient recruitment
Further sampling timepoints will be immediately post-op T1 24h post-op T2 discharge T3 and 4 weeks post-op at surgical follow up T4 The blood collected at 4-week post-surgery follow up will be assayed for IgG antibody assay to EndoCAb α-toxin and teichoic acid This will enable a measure of class switch recombination Which will be referable back to the degree of expression of AID mRNA
The population of aortic valve replacement patients will be followed up for mortality and major morbidity They will be scored as per C-POMS cardiac post-operative morbidity score length of ITU stay and length of hospital stay Patients will be monitored for development of surgical site infection SSI or the acquisition of post-operative hospital acquired infection HAI Post-discharge follow-up for wound infection or breakdown will be continued for 12 months
At the immediately pre-operative timepoint T0 alongside currently accepted measures of immune responsiveness it is intended to perform a near patient test of immune responsiveness LIT using a handheld chemiluminescence device that relies upon leukocyte oxidative burst to a supramaximal stimulus Oxford Medistress This relies upon analysis of the response in a 10mcl fresh capillary blood sample
Statistical considerations
From published data from centres worldwide it is known that a proportion of patients develop an infection either surgical site infection SSI or hospital acquired infection HAI following aortic valve surgery It is also known that these patients endure longer stay in hospital following surgery
The investigators have demonstrated that amongst these patients there is variability in levels of antibodies to various perioperative threats namely endotoxin and staphylococcus
The investigators have demonstrated that in the patients who are undergoing surgery and have low levels of circulating antibody to endotoxin andor staphylococcus there is an association with greater likelihood of developing an infection and a longer length of post-operative stay The investigators estimate that the rate of infection varies between 135 - 33
The investigators have previously suggested that the association between antibody level and outcome may be manifestation of an underlying impaired ability in some individuals to mount a normal immune response to these threats
It is known that a proportion of individuals have an abnormal immune response This is perhaps most obviously seen in the response to vaccination where previous work has demonstrated the lack of immune responsiveness following various vaccination programs and our own work on EndoCAb response to monovalent typhoid vaccination This nonpoor responsiveness in these studies lies somewhere between 10 and 40 The diagram illustrates the proportion of non-responders ie poor immune responsiveness if they are equally distributed
Setting
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 QMULWHRI 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 20C and plasma stored at -80C 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 LPSα-toxin Stimulated Cytokine Release
Technique As previously described and validated in our laboratory heparinized blood will be stimulated for 4hrs 37C 250rpm with 1ngml LPS within 2hrs of draw After incubation samples will be centrifuged 1200g 10mins 20C and supernatant stored at -80C
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 CST 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 105
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
MonocyteMyeloid 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 Ecoli bacteria will be quantified via the PhagoTest assay BD as per manufacturers instructions Reactive oxygen burst Quantitative determination of leukocyte oxidative burst will be undertaken using the PhagoBurst assay BD in response to un-labelled opsonized Ecoli phorbol 12-myristate 13-acetate PMA and the chemotactic peptide N-formyl-Met-Leu-Phe fMLP
o 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 qPCR 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
Enough healthy volunteer blood is available so that all above techniques can be adequately practiced and optimized prior to patient recruitment
Further sampling timepoints will be immediately post-op T1 24h post-op T2 discharge T3 and 4 weeks post-op at surgical follow up T4 The blood collected at 4-week post-surgery follow up will be assayed for IgG antibody assay to EndoCAb α-toxin and teichoic acid This will enable a measure of class switch recombination Which will be referable back to the degree of expression of AID mRNA
The population of aortic valve replacement patients will be followed up for mortality and major morbidity They will be scored as per C-POMS cardiac post-operative morbidity score length of ITU stay and length of hospital stay Patients will be monitored for development of surgical site infection SSI or the acquisition of post-operative hospital acquired infection HAI Post-discharge follow-up for wound infection or breakdown will be continued for 12 months
At the immediately pre-operative timepoint T0 alongside currently accepted measures of immune responsiveness it is intended to perform a near patient test of immune responsiveness LIT using a handheld chemiluminescence device that relies upon leukocyte oxidative burst to a supramaximal stimulus Oxford Medistress This relies upon analysis of the response in a 10mcl fresh capillary blood sample
Statistical considerations
From published data from centres worldwide it is known that a proportion of patients develop an infection either surgical site infection SSI or hospital acquired infection HAI following aortic valve surgery It is also known that these patients endure longer stay in hospital following surgery
The investigators have demonstrated that amongst these patients there is variability in levels of antibodies to various perioperative threats namely endotoxin and staphylococcus
The investigators have demonstrated that in the patients who are undergoing surgery and have low levels of circulating antibody to endotoxin andor staphylococcus there is an association with greater likelihood of developing an infection and a longer length of post-operative stay The investigators estimate that the rate of infection varies between 135 - 33
The investigators have previously suggested that the association between antibody level and outcome may be manifestation of an underlying impaired ability in some individuals to mount a normal immune response to these threats
It is known that a proportion of individuals have an abnormal immune response This is perhaps most obviously seen in the response to vaccination where previous work has demonstrated the lack of immune responsiveness following various vaccination programs and our own work on EndoCAb response to monovalent typhoid vaccination This nonpoor responsiveness in these studies lies somewhere between 10 and 40 The diagram illustrates the proportion of non-responders ie poor immune responsiveness if they are equally distributed
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None