<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT04430972</url>
  </required_header>
  <id_info>
    <org_study_id>263703</org_study_id>
    <nct_id>NCT04430972</nct_id>
  </id_info>
  <brief_title>Immune Responsiveness and Outcome After Aortic Valve Surgery (Measure)</brief_title>
  <acronym>Measure</acronym>
  <official_title>Is Pre-operative Impaired imMune rEsponsiveness Associated With Adverse Outcome Following Aortic Valve Replacement SURgEry (MEASURE)</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>Barts &amp; The London NHS Trust</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
    <collaborator>
      <agency>University College, London</agency>
      <agency_class>Other</agency_class>
    </collaborator>
  </sponsors>
  <source>Barts &amp; The London NHS Trust</source>
  <oversight_info>
    <has_dmc>No</has_dmc>
    <is_fda_regulated_drug>No</is_fda_regulated_drug>
    <is_fda_regulated_device>No</is_fda_regulated_device>
  </oversight_info>
  <brief_summary>
    <textblock>
      There is considerable morbidity and mortality associated with cardiac surgery. Currently&#xD;
      little effort is made to quantify how well the immune system of an individual can cope with&#xD;
      inflammation or infection to which they are exposed during surgery.&#xD;
&#xD;
      The investigators have previously demonstrated that having higher pre-operative antibody&#xD;
      levels is associated with a lower risk of infection and a shorter stay in hospital after&#xD;
      cardiac surgery.&#xD;
&#xD;
      The investigators aim to study 150 patients undergoing aortic valve replacement and explore&#xD;
      their dynamic immune responsiveness. The investigators will determine if this response is&#xD;
      correlated with the post-operative outcome (development of post-operative infection or&#xD;
      increased length of hospital stay).&#xD;
&#xD;
      The investigators will compare this response with the previously measured static markers of&#xD;
      immune competence and also with a novel device that may give a more rapid measure of dynamic&#xD;
      immunity.&#xD;
&#xD;
      The investigators will approach patients in the cardiac surgical pre-assessment clinic to see&#xD;
      if they are willing to participate in the study.&#xD;
&#xD;
      Immediately once under anaesthetic blood will be taken for testing and then again at the end&#xD;
      of surgery, 24h after surgery, at discharge from hospital, and at follow-up clinic&#xD;
      approximately 4 weeks later. There will be no additional needle insertions on top of those&#xD;
      routinely performed. The investigators will collect data from the routine observations as far&#xD;
      as 1 year after surgery.&#xD;
&#xD;
      If the investigators can show an association between immune function and subsequent&#xD;
      post-operative outcome it may be possible to determine ways to improve outcomes for patients&#xD;
      undergoing heart surgery.&#xD;
&#xD;
      This might include better information on risks and benefits of surgery, actively boosting&#xD;
      immune function (vaccination, immune-nutrition), passively improving immunity (administering&#xD;
      antibodies), or consider current alternatives to open heart surgery where the threat of&#xD;
      infection or inflammation may be markedly reduced (eg trans-catheter aortic valve&#xD;
      implantation)&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      Experimental design and methods:&#xD;
&#xD;
      Setting:&#xD;
&#xD;
      Barts Heart Centre is a 255-bedded specialist cardiac centre where approximately 200 primary&#xD;
      isolated open aortic valve replacement procedures are performed per year. It is closely&#xD;
      associated both geographically and academically with the William Harvey Research Institute&#xD;
      (WHRI), which forms part of Queen Mary University, London (QMUL). This will allow laboratory&#xD;
      analysis of all collected blood samples within 15 minutes if necessary. Barts Heart Centre is&#xD;
      one of the largest cardiac units in Europe and a key research objective of QMUL/WHRI is to&#xD;
      support cardiac research.&#xD;
&#xD;
      Sample collection and storage:&#xD;
&#xD;
      Following written informed consent all patients will be pre-operatively risk-scored by means&#xD;
      of ASA, Euroscore 2 and conventionally risk-scored for the development of surgical site&#xD;
      infection.&#xD;
&#xD;
      Blood (44ml total) will be taken from 150 aortic valve replacement patients immediately prior&#xD;
      to surgery (T0).&#xD;
&#xD;
      Blood (7ml) will be collected into a PAXgene tube and stored at -80° for later genetic&#xD;
      analysis. A clotted sample (7ml) will be collected in a standard gold top SST tube, left to&#xD;
      clot then centrifuged for 10 mins at 1000g, the serum separated and stored at -80° for later&#xD;
      analysis of antibody levels. In addition, 30 ml blood will be taken for study of peripheral&#xD;
      blood mononuclear cells (PBMCs), drawn into standard purple top EDTA tubes (anticoagulant).&#xD;
      These will be separated within 3 hours of collection by Ficoll-Paque density gradient&#xD;
      centrifugation, whereby PBS-diluted blood is carefully layered over Ficoll and spun brake off&#xD;
      in a centrifuge at 400g for 30 minutes. The PBMC layer is then extracted and washed twice in&#xD;
      PBS and stored in 10% dimethyl sulfoxide freezing solution in liquid nitrogen so that they&#xD;
      can be thawed, and batch analysed at a later date.&#xD;
&#xD;
      Sample analysis:&#xD;
&#xD;
      PBMCs will be counted using a haemocytometer, then cultured in RPMI and 10% serum with either&#xD;
      LPS, α-toxin or unstimulated for 24 hours, after which they will be analysed immediately as&#xD;
      detailed below.&#xD;
&#xD;
      • Soluble Mediators (Plasma)&#xD;
&#xD;
        -  Storage: Ethylenediaminetetraacetic acid (EDTA, BD Biosciences Vacutainer, 9mL)&#xD;
           anticoagulated blood will be centrifuged (1200g, 10mins, 20°C) and plasma stored at&#xD;
           -80°C within 2hrs of collection&#xD;
&#xD;
        -  Analysis:&#xD;
&#xD;
      Cytokines: Meso Scale Discovery (MSD) V-PLEX Proinflammatory Panel 1 will be employed to&#xD;
      quantify IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, and TNF-α. Plates will&#xD;
      be read using a MSD QuickPlex SQ 120 imager (Wiliam Harvey Institute, QMUL).&#xD;
&#xD;
      Antibodies: These will be measured by means of ELISA assays against staphylococcal antigens&#xD;
      (α-toxin, teichoic acid) and core moieties of the endotoxin molecule (EndoCAb).&#xD;
&#xD;
        -  Whole blood LPS/α-toxin Stimulated Cytokine Release&#xD;
&#xD;
             -  Technique: As previously described and validated in our laboratory, heparinized&#xD;
                blood will be stimulated for 4hrs (37°C, 250rpm) with 1ng/ml LPS within 2hrs of&#xD;
                draw. After incubation, samples will be centrifuged (1200g, 10mins, 20°C) and&#xD;
                supernatant stored at -80°C.&#xD;
&#xD;
             -  Cytokine quantification: As per 'soluble mediators' MSD V-PLEX Proinflammatory&#xD;
                Panel 1 will be employed to quantify TNF-α (primary outcome as metric of immune&#xD;
                competence) and other cytokines. Cytokines will be expressed as a factor of the&#xD;
                number of circulating monocytes.&#xD;
&#xD;
        -  Flow Cytometric Immunophenotyping and Functional Assessment of Leukocytes&#xD;
&#xD;
             -  Flow Cytometry: All samples will be analysed on the same machine (Becton Dickinson&#xD;
                (BD) Fortessa, Rayne Building, UCL). Standardisation and comparison of output will&#xD;
                be achieved via employment of constant voltages and compensation matrix throughout&#xD;
                with daily matching of values to a single batch of Cytometry Setup and Tracking&#xD;
                (CS&amp;T) beads (BD). Leukocyte cell surface staining will be conducted using&#xD;
                antibodies principally supplied by BD. Staining, data capture and storage will be&#xD;
                conducted in accordance with a single study standard operating procedure. 5 panels&#xD;
                (up to 14-colour) and two functional assays will be performed at each time-point.&#xD;
                Data will be analysed in FlowJo version 10.5.&#xD;
&#xD;
             -  Surface Marker Staining:&#xD;
&#xD;
      Quantification and calibration panel: CD45, CD56, CD3, CD4, CD8, CD19, CD14, CD16, performed&#xD;
      in a BD TruCount tube to enable enumeration of cell subsets Cell specific panels&#xD;
      (incorporating markers of sub-categorisation and markers of immune competence)&#xD;
&#xD;
        -  Monocyte/Myeloid: CD3, CD19, CD56, CD163, CD16, CD33, CD141, CD206, CD274, CD14, CD1c,&#xD;
           CD11c, HLA-DR, CD80, CD123, CD83&#xD;
&#xD;
        -  Neutrophil: CD3, CD19, CD56, HLA-DR, CD64, CD62L, CD11b, CD88, CD66b, CD14, CD11c, CD16,&#xD;
           CD184, CD182&#xD;
&#xD;
        -  Lymphocyte: CD19, CCR6, CD45RA, CD3, CD4, CD8, CXCR3, CD127, CD62L, CD25, CD56, CD27,&#xD;
           CD279 HLA-DR Expression: Will be quantified on CD14+ monocytes using BD QuantiBrite&#xD;
           beads.&#xD;
&#xD;
             -  Functional Measures Phagocytosis: The ability of neutrophils and monocytes to&#xD;
                phagocytose opsonised FITC-labelled E.coli bacteria will be quantified via the&#xD;
                PhagoTest assay (BD) as per manufacturer's instructions Reactive oxygen 'burst':&#xD;
                Quantitative determination of leukocyte oxidative burst will be undertaken using&#xD;
                the PhagoBurst assay (BD) in response to un-labelled opsonized E.coli, phorbol&#xD;
                12-myristate 13-acetate (PMA) and the chemotactic peptide N-formyl-Met-Leu-Phe&#xD;
                (fMLP).&#xD;
&#xD;
      o The pre-surgery sample PBMCs that will be stimulated with LPS (type and concentration to be&#xD;
      optimized via dose response curve on healthy volunteers) and α-toxin will undergo flow&#xD;
      cytometry to measure TLR4 and TLR5 surface expression, HLA-DR surface expression and&#xD;
      quantification of NF-kB using appropriate antibodies validated in the literature. At the end&#xD;
      of stimulation, cells will be harvested, and RNA extracted for quantitative (q)PCR to&#xD;
      evaluate AID mRNA expression. Although B cells in the PBMC cultures have been stimulated in&#xD;
      the presence of other cell types, primarily T cells and monocytes-macrophages, our endpoint&#xD;
      is to measure a B-cell response, as AID is exclusively expressed in B cells.&#xD;
&#xD;
      Enough healthy volunteer blood is available so that all above techniques can be adequately&#xD;
      practiced and optimized prior to patient recruitment.&#xD;
&#xD;
      Further sampling timepoints will be immediately post-op (T1), 24h post-op (T2), discharge&#xD;
      (T3) and 4 weeks post-op at surgical follow up (T4). The blood collected at 4-week&#xD;
      post-surgery follow up will be assayed for IgG antibody assay to EndoCAb, α-toxin and&#xD;
      teichoic acid. This will enable a measure of class switch recombination. Which will be&#xD;
      referable back to the degree of expression of AID mRNA.&#xD;
&#xD;
      The population of aortic valve replacement patients will be followed up for mortality and&#xD;
      major morbidity. They will be scored as per C-POMS (cardiac post-operative morbidity score),&#xD;
      length of ITU stay and length of hospital stay. Patients will be monitored for development of&#xD;
      surgical site infection (SSI) or the acquisition of post-operative hospital acquired&#xD;
      infection (HAI). Post-discharge follow-up for wound infection or breakdown will be continued&#xD;
      for 12 months.&#xD;
&#xD;
      At the immediately pre-operative timepoint (T0), alongside currently accepted measures of&#xD;
      immune responsiveness it is intended to perform a near patient test of immune responsiveness&#xD;
      (LIT) using a handheld chemiluminescence device that relies upon leukocyte oxidative burst to&#xD;
      a supramaximal stimulus (Oxford Medistress). This relies upon analysis of the response in a&#xD;
      10mcl fresh capillary blood sample.&#xD;
&#xD;
      Statistical considerations:&#xD;
&#xD;
      From published data from centres worldwide it is known that a proportion of patients develop&#xD;
      an infection, either surgical site infection (SSI) or hospital acquired infection (HAI),&#xD;
      following aortic valve surgery. It is also known that these patients endure longer stay in&#xD;
      hospital following surgery.&#xD;
&#xD;
      The investigators have demonstrated that amongst these patients there is variability in&#xD;
      levels of antibodies to various perioperative threats, namely endotoxin and staphylococcus.&#xD;
&#xD;
      The investigators have demonstrated that in the patients who are undergoing surgery and have&#xD;
      low levels of circulating antibody to endotoxin and/or staphylococcus there is an association&#xD;
      with greater likelihood of developing an infection and a longer length of post-operative&#xD;
      stay. The investigators estimate that the rate of infection varies between 13.5 - 33%.&#xD;
&#xD;
      The investigators have previously suggested that the association between antibody level and&#xD;
      outcome may be manifestation of an underlying impaired ability in some individuals to mount a&#xD;
      normal immune response to these threats.&#xD;
&#xD;
      It is known that a proportion of individuals have an abnormal immune response. This is&#xD;
      perhaps most obviously seen in the response to vaccination where previous work has&#xD;
      demonstrated the lack of immune responsiveness following various vaccination programs and our&#xD;
      own work on EndoCAb response to monovalent typhoid vaccination. This non/poor responsiveness&#xD;
      in these studies lies somewhere between 10 and 40%. The diagram illustrates the proportion of&#xD;
      non-responders (ie poor immune responsiveness) if they are equally distributed.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Not yet recruiting</overall_status>
  <start_date type="Anticipated">September 2023</start_date>
  <completion_date type="Anticipated">September 2026</completion_date>
  <primary_completion_date type="Anticipated">September 2025</primary_completion_date>
  <study_type>Observational</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Cohort</observational_model>
    <time_perspective>Prospective</time_perspective>
  </study_design_info>
  <primary_outcome>
    <measure>Number of participants who develop any post operative, healthcare associated infection.</measure>
    <time_frame>Development of a HealthCare Associated Infection (HCAI) will be evaluated at one year from date of discharge from hospital.</time_frame>
    <description>Development of post-operative healthcare associated infection as defined in the Centre for Disease Control and National Healthcare Safety Network guidance.</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Post-operative length of stay</measure>
    <time_frame>Will be assessed at study completion, at one year following discharge from hospital.</time_frame>
    <description>Time spent in hospital following surgery measured in days</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Correlation of pre-operative antibody levels with in vitro immune response profiles</measure>
    <time_frame>2 years</time_frame>
    <description>Determining if there is a correlation between those patients with low antibody levels pre-operatively to endotoxin core and staphylococcal epitopes with the dynamic immune responsiveness in an in vitro test of immune response</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Does the result from a simple, point of care test of immune function correlate with that from a more comprehensive, laboratory based test of immune function</measure>
    <time_frame>2 years</time_frame>
    <description>To determine whether the result obtained from a bedside test of immune function (LITTM) correlates with the result obtained from an in-vitro, laboratory-based test of immune function.&#xD;
The bedside test relies on the chemiluminescence result from a supramaximal stimulus of neutrophils and gives a result in the form of a continuous variable, allowing the ranking of the subjects in order of their neutrophil responsiveness.&#xD;
The laboratory-based tests, performed using flow cytometry, will look at the immune function of monocytes and each assay of a number of cytokines and cell markers will give results as a continuous variable. This again will allow for an overall ranking of patients in terms of the value obtained for each parameter measured. Participants will also be ranked according to the summed rank positions for each variable.&#xD;
Correlation between the rank order of patients determined by the two separate assays of function will be compared by calculating correlation coefficient.</description>
  </secondary_outcome>
  <enrollment type="Anticipated">150</enrollment>
  <condition>Aortic Valve Disease</condition>
  <condition>Surgery--Complications</condition>
  <condition>Immune System Disorder</condition>
  <intervention>
    <intervention_type>Procedure</intervention_type>
    <intervention_name>Aortic valve replacement</intervention_name>
    <description>Open surgical replacement of aortic valve</description>
  </intervention>
  <biospec_retention>Samples With DNA</biospec_retention>
  <biospec_descr>
    <textblock>
      Serum Plasma PaxGene for genetic sequencing Monocytes&#xD;
    </textblock>
  </biospec_descr>
  <eligibility>
    <study_pop>
      <textblock>
        All adult patients undergoing first-time aortic valve replacement that do not fulfil any of&#xD;
        the exclusion criteria&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Non-Probability Sample</sampling_method>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
        • Patients undergoing first-time open aortic valve replacement surgery&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  Age less than 18y (capacity to consent)&#xD;
&#xD;
          -  Ongoing sepsis (immunomodulatory effects, established influence on outcome from&#xD;
             surgery)&#xD;
&#xD;
          -  Immunosuppressive therapy&#xD;
&#xD;
          -  Suffering from known immunosuppressive disease&#xD;
&#xD;
          -  Pregnancy (immunosuppressive aspects to pregnancy)&#xD;
      </textblock>
    </criteria>
    <gender>All</gender>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>N/A</maximum_age>
    <healthy_volunteers>No</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>Chris Thiemermann</last_name>
    <role>Study Director</role>
    <affiliation>Queen Mary University London</affiliation>
  </overall_official>
  <overall_contact>
    <last_name>Colin Hamilton-Davies, MBBS MD</last_name>
    <phone>+44(0)2037658348</phone>
    <email>colin.hamilton-davies@qmul.ac.uk</email>
  </overall_contact>
  <overall_contact_backup>
    <last_name>Robert Smith, MBBS FRCA</last_name>
    <phone>+44(0)2037658348</phone>
    <email>andrew.smith@qmul.ac.uk</email>
  </overall_contact_backup>
  <location>
    <facility>
      <name>St Bartholomew's Hospital</name>
      <address>
        <city>London</city>
        <zip>EC1A 7BE</zip>
        <country>United Kingdom</country>
      </address>
    </facility>
    <contact>
      <last_name>Colin Hamilton-Davies</last_name>
      <email>colin.hamilton-davies@qmul.ac.uk</email>
    </contact>
    <contact_backup>
      <last_name>Robert A Smith</last_name>
      <email>andrew.smith@qmul.ac.uk</email>
    </contact_backup>
  </location>
  <location_countries>
    <country>United Kingdom</country>
  </location_countries>
  <results_reference>
    <citation>Smith A, Moravcova S, Treibel TA, Colque-Navarro P, Mollby R, Moon JC, Hamilton-Davies C. Relationship between endotoxin core, staphylococcal and varicella antibody levels and outcome following aortic valve replacement surgery: a prospective observational study. Perioper Med (Lond). 2018 Sep 20;7:20. doi: 10.1186/s13741-018-0101-z. eCollection 2018.</citation>
    <PMID>30250733</PMID>
  </results_reference>
  <results_reference>
    <citation>Moravcova S, Kyle B, Shanahan H, Giannaris S, Smith A, Hamilton-Davies C. Variability of anti-staphylococcal antibodies in healthy volunteers and pre-cardiac surgery patients. Perioper Med (Lond). 2016 May 27;5:13. doi: 10.1186/s13741-016-0039-y. eCollection 2016.</citation>
    <PMID>27239299</PMID>
  </results_reference>
  <results_reference>
    <citation>Hamilton-Davies C, Barclay GR, Cardigan RA, McDonald SJ, Purdy G, Machin SJ, Webb AR. Relationship between preoperative endotoxin immune status, gut perfusion, and outcome from cardiac valve replacement surgery. Chest. 1997 Nov 5;112(5):1189-96. doi: 10.1378/chest.112.5.1189.</citation>
    <PMID>9367456</PMID>
  </results_reference>
  <results_reference>
    <citation>Bennett-Guerrero E, Ayuso L, Hamilton-Davies C, White WD, Barclay GR, Smith PK, King SA, Muhlbaier LH, Newman MF, Mythen MG. Relationship of preoperative antiendotoxin core antibodies and adverse outcomes following cardiac surgery. JAMA. 1997 Feb 26;277(8):646-50.</citation>
    <PMID>9039883</PMID>
  </results_reference>
  <results_reference>
    <citation>Hamilton-Davies C, Barclay GR, Murphy WG, Machin SJ, Webb AR. Passive immunisation with IgG endotoxin core antibody hyperimmune fresh frozen plasma. Vox Sang. 1996;71(3):165-9. doi: 10.1046/j.1423-0410.1996.7130165.x.</citation>
    <PMID>8912459</PMID>
  </results_reference>
  <results_reference>
    <citation>Poland GA, Ovsyannikova IG, Jacobson RM. Vaccine immunogenetics: bedside to bench to population. Vaccine. 2008 Nov 18;26(49):6183-8. doi: 10.1016/j.vaccine.2008.06.057. Epub 2008 Jul 1.</citation>
    <PMID>18598732</PMID>
  </results_reference>
  <verification_date>April 2022</verification_date>
  <study_first_submitted>May 20, 2020</study_first_submitted>
  <study_first_submitted_qc>June 11, 2020</study_first_submitted_qc>
  <study_first_posted type="Actual">June 16, 2020</study_first_posted>
  <last_update_submitted>January 25, 2023</last_update_submitted>
  <last_update_submitted_qc>January 25, 2023</last_update_submitted_qc>
  <last_update_posted type="Actual">January 26, 2023</last_update_posted>
  <responsible_party>
    <responsible_party_type>Sponsor</responsible_party_type>
  </responsible_party>
  <keyword>staphylococcus</keyword>
  <keyword>EndoCAb</keyword>
  <keyword>Immunity</keyword>
  <keyword>Cardiac Surgical Procedure</keyword>
  <keyword>sepsis</keyword>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Aortic Valve Disease</mesh_term>
    <mesh_term>Immune System Diseases</mesh_term>
  </condition_browse>
  <patient_data>
    <sharing_ipd>Undecided</sharing_ipd>
  </patient_data>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT04430972
org study id: 263703
nct id: NCT04430972
lead sponsor: 
collaborator: 
has dmc: No
is fda regulated drug: No
is fda regulated device: No
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)
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 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).

      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 (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.

      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 and/or 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 13.5 - 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 non/poor 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.
observational model: Cohort
time perspective: Prospective
measure: Number of participants who develop any post operative, healthcare associated infection.
time frame: Development of a HealthCare Associated Infection (HCAI) will be evaluated at one year from date of discharge from hospital.
description: Development of post-operative healthcare associated infection as defined in the Centre for Disease Control and National Healthcare Safety Network guidance.
measure: Post-operative length of stay
time frame: Will be assessed at study completion, at one year following discharge from hospital.
description: Time spent in hospital following surgery measured in days
measure: Correlation of pre-operative antibody levels with in vitro immune response profiles
time frame: 2 years
description: Determining if there is a correlation between those patients with low antibody levels pre-operatively to endotoxin core and staphylococcal epitopes with the dynamic immune responsiveness in an in vitro test of immune response
measure: Does the result from a simple, point of care test of immune function correlate with that from a more comprehensive, laboratory based test of immune function
time frame: 2 years
description: To determine whether the result obtained from a bedside test of immune function (LITTM) correlates with the result obtained from an in-vitro, laboratory-based test of immune function.
The bedside test relies on the chemiluminescence result from a supramaximal stimulus of neutrophils and gives a result in the form of a continuous variable, allowing the ranking of the subjects in order of their neutrophil responsiveness.
The laboratory-based tests, performed using flow cytometry, will look at the immune function of monocytes and each assay of a number of cytokines and cell markers will give results as a continuous variable. This again will allow for an overall ranking of patients in terms of the value obtained for each parameter measured. Participants will also be ranked according to the summed rank positions for each variable.
Correlation between the rank order of patients determined by the two separate assays of function will be compared by calculating correlation coefficient.
intervention type: Procedure
intervention name: Aortic valve replacement
description: Open surgical replacement of aortic valve
Serum Plasma PaxGene for genetic sequencing Monocytes
study pop: 
sampling method: Non-Probability Sample
criteria: 
gender: All
minimum age: 18 Years
maximum age: N/A
healthy volunteers: No
last name: Chris Thiemermann
role: Study Director
affiliation: Queen Mary University London
last name: Colin Hamilton-Davies, MBBS MD
phone: +44(0)2037658348
email: colin.hamilton-davies@qmul.ac.uk
last name: Robert Smith, MBBS FRCA
phone: +44(0)2037658348
email: andrew.smith@qmul.ac.uk
facility: 
contact: 
contact backup: 
country: United Kingdom
citation: Smith A, Moravcova S, Treibel TA, Colque-Navarro P, Mollby R, Moon JC, Hamilton-Davies C. Relationship between endotoxin core, staphylococcal and varicella antibody levels and outcome following aortic valve replacement surgery: a prospective observational study. Perioper Med (Lond). 2018 Sep 20;7:20. doi: 10.1186/s13741-018-0101-z. eCollection 2018.
PMID: 30250733
citation: Moravcova S, Kyle B, Shanahan H, Giannaris S, Smith A, Hamilton-Davies C. Variability of anti-staphylococcal antibodies in healthy volunteers and pre-cardiac surgery patients. Perioper Med (Lond). 2016 May 27;5:13. doi: 10.1186/s13741-016-0039-y. eCollection 2016.
PMID: 27239299
citation: Hamilton-Davies C, Barclay GR, Cardigan RA, McDonald SJ, Purdy G, Machin SJ, Webb AR. Relationship between preoperative endotoxin immune status, gut perfusion, and outcome from cardiac valve replacement surgery. Chest. 1997 Nov 5;112(5):1189-96. doi: 10.1378/chest.112.5.1189.
PMID: 9367456
citation: Bennett-Guerrero E, Ayuso L, Hamilton-Davies C, White WD, Barclay GR, Smith PK, King SA, Muhlbaier LH, Newman MF, Mythen MG. Relationship of preoperative antiendotoxin core antibodies and adverse outcomes following cardiac surgery. JAMA. 1997 Feb 26;277(8):646-50.
PMID: 9039883
citation: Hamilton-Davies C, Barclay GR, Murphy WG, Machin SJ, Webb AR. Passive immunisation with IgG endotoxin core antibody hyperimmune fresh frozen plasma. Vox Sang. 1996;71(3):165-9. doi: 10.1046/j.1423-0410.1996.7130165.x.
PMID: 8912459
citation: Poland GA, Ovsyannikova IG, Jacobson RM. Vaccine immunogenetics: bedside to bench to population. Vaccine. 2008 Nov 18;26(49):6183-8. doi: 10.1016/j.vaccine.2008.06.057. Epub 2008 Jul 1.
PMID: 18598732
responsible party type: Sponsor
mesh term: Aortic Valve Disease
mesh term: Immune System Diseases
sharing ipd: Undecided