Prevention of Myocardial Injury by Remote Ischemic Preconditioning in Emergent or Urgent Non-cardiac Surgery: a Randomized Clinical Trial
Phase: NA
Conditions: Myocardial Injury, Myocardial Infarction
Sponsor: Zealand University Hospital

 
 
 
 
Prevention of myocardial injury by remote ischemic preconditioning in 
emergent or urgent non-cardiac surgery: a randomized clinical trial 
The PIXIE study 
 
This supplement contains the following items: 
1. Original protocol, final protocol, summary of changes (protocol). 
2. Original statistical analysis plan, final statistical analysis plan, summary of changes   
(statistical analysis plan). 
 
 

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Version	  3.4	  
	  
Prevention	  of	  myocardial	  injury	  by	  remote	  ischemic	  
preconditioning	  in	  emergent	  or	  urgent	  non-­‐cardiac	  
surgery:	  a	  randomized	  clinical	  trial	  
The	  PIXIE	  study	  
	  
Contact	  information	  
Primary	  investigator:	  Sarah	  Ekeløf	  Busch,	  MD,	  Ph.D.-­‐student,	  Department	  of	  Surgery,	  Køge	  
Hospital,	  Lykkebækvej	  1,	  4600	  Køge.	  	  
Sponsor,	  investigator:	  Ismail	  Gögenur,	  Professor,	  MD,	  DMSc,	  Department	  of	  Surgery,	  Køge	  
Hospital,	  Lykkebækvej	  1,	  4600	  Køge.	  	  
Investigator:	  Ole	  Mathiesen,	  MD,	  Ph.D.,	  Department	  of	  Anesthesiology,	  Køge	  Hospital,	  
Lykkebækvej	  1,	  4600	  Køge.	  	  
Investigator:	  Mikkel	  Holm	  Larsen,	  medical	  student,	  Department	  of	  Surgery,	  Køge	  Hospital,	  
Lykkebækvej	  1,	  4600	  Køge.	  	  
	  
Applicant:	  Sarah	  Ekeløf	  Busch,	  MD,	  Ph.D.-­‐student,	  Department	  of	  Surgery,	  Køge	  Hospital,	  
Lykkebækvej	  1,	  4600	  Køge.	  
Tlf:	  +45	  22	  71	  28	  74	  
E-­‐mail:	  sarahekeloef@gmail.com	  	  
	  
Signature/Date:	  
 / 20.11.2014 
Sarah Ekeløf Busch 
Applicant,	  Sarah	  Ekeløf	  Busch	  
	  
Introduction	  	  

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Worldwide,	  more	  than	  200	  million	  patients	  have	  major	  non-­‐cardiac	  surgery	  annually	  and	  a	  
significant	  proportion	  of	  these	  patients	  suffer	  major	  cardiovascular	  complications	  (e.g.	  
nonfatal	  myocardial	  infarction,	  cardiac	  arrest,	  vascular	  death)	  within	  30	  days	  of	  their	  surgery	  
[1,	  2].	  Perioperative	  myocardial	  infarction	  is	  the	  most	  common	  cardiovascular	  complication	  
and	  recent	  clinical	  studies	  have	  shown	  that	  even	  minor	  myocardial	  injury	  in	  relation	  to	  non-­‐
cardiac	  surgery	  is	  associated	  with	  30-­‐day	  mortality	  [3,	  4].	  	  
The	  pathogenesis	  of	  perioperative	  myocardial	  infarction	  is	  complex	  and	  not	  fully	  
elucidated.	  Surgery	  is	  associated	  with	  trauma,	  anesthesia	  and	  analgesia,	  bleeding	  and	  anemia,	  
pain,	  hypothermia	  and	  fasting	  [2].	  These	  factors	  initiate	  systemic	  inflammation,	  
hypercoagulability,	  increased	  levels	  of	  catecholamines	  and	  cortisol	  and	  a	  state	  of	  hypoxia	  [2].	  
The	  drastic	  systemic	  changes	  can	  lead	  to	  a	  myocardial	  hypoxic	  supply-­‐demand	  mismatch	  
where	  rupture	  of	  vulnerable	  coronary	  plaques	  might	  represent	  an	  additional	  mechanism	  [2,	  
5].	  At	  the	  moment,	  no	  effective	  prevention	  or	  treatment	  of	  perioperative	  myocardial	  
infarction	  in	  non-­‐cardiac	  surgery	  is	  accepted.	  	  
The	  aim	  of	  this	  study	  is	  to	  determine	  whether	  an	  intervention	  with	  remote	  ischemic	  
preconditioning	  can	  reduce	  myocardial	  injury	  in	  emergent	  or	  urgent	  non-­‐cardiac	  surgery.	  	  
	  
Remote	  ischemic	  preconditioning	  
Remote	  ischemic	  preconditioning	  is	  a	  procedure,	  which	  protects	  remote	  tissues	  and	  
organs	  e.g.	  against	  ischemia-­‐reperfusion	  injury	  [6].	  Cycles	  of	  forearm	  or	  leg	  ischemia	  and	  
reperfusion	  by	  the	  inflation	  of	  a	  blood-­‐pressure	  cuff	  for	  brief	  periods	  are	  the	  preferred	  
method	  [7].	  The	  procedure	  is	  simple,	  safe	  and	  with	  no	  clear	  side	  effects	  [6].	  	  
The	  mechanism	  of	  remote	  ischemic	  preconditioning	  is	  not	  fully	  understood.	  However,	  it	  
seems	  that	  the	  procedure	  triggers	  complex	  endogenous	  pathways	  including	  a	  humoral	  
pathway,	  a	  neural	  pathway	  and	  a	  systemic	  anti-­‐inflammatory	  pathway	  [7,	  8].	  	  
In	  clinical	  trials	  covering	  acute	  cardiology	  and	  cardiac	  surgery,	  remote	  ischemic	  
preconditioning	  has	  effectively	  reduced	  myocardial	  injury,	  postoperative	  cardiovascular	  
complications	  and	  cardiac	  mortality	  [9,	  10].	  Recently,	  the	  effect	  of	  remote	  ischemic	  
preconditioning	  on	  attenuating	  ischemia-­‐reperfusion	  injury	  has	  been	  investigated	  in	  non-­‐
cardiac	  surgery	  [11-­‐14].	  The	  organ	  specific	  ischemia-­‐reperfusion	  injury,	  systemic	  oxidative	  
stress	  and	  inflammatory	  response	  were	  attenuated	  due	  to	  the	  intervention	  [11-­‐14].	  Whether	  

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remote	  ischemic	  preconditioning	  protects	  against	  myocardial	  injury	  in	  the	  perioperative	  
period	  of	  non-­‐cardiac	  surgery	  have	  until	  now	  not	  been	  investigated.	  	  
	  
Aim	  
- 
To	  determine	  whether	  remote	  ischemic	  preconditioning	  can	  reduce	  markers	  of	  
myocardial	  injury	  in	  emergent	  or	  urgent	  non-­‐cardiac	  surgery.	   
	  
Methods	  
Trial	  design	  
Randomized	  clinical	  trial	  	  	  	  
	  
Study	  setting	  
Department	  of	  Surgery,	  Køge	  Hospital,	  Denmark	  
Department	  of	  Orthopedic	  Surgery,	  Køge	  Hospital,	  Denmark	  
Department	  of	  Anesthesia,	  Køge	  Hospital,	  Denmark	  
	  
Eligibility	  criteria	  
Inclusion	  criteria	  
▪ patients	  ≥	  45	  years	  undergoing	  in-­‐hospital	  major	  non-­‐cardiac	  surgery	   
▪ major	  general	  surgery	   
• 
surgery	  with	  planned	  laparotomy	  or	  high	  risk	  of	  laparotomy	  or	  
perforated	  intraperitoneal	  organ	  except	  the	  appendix.	   
▪ major	  orthopedic	  surgery	   
• 
hip	  surgery	   
▪ undergoing	  emergent	  or	  urgent	  surgery	  (i.e.	  emergent	  or	  urgent	  visitation	  to	  the	  
Department	  of	  Surgery	  or	  Department	  of	  Orthopedic	  Surgery) 
▪ fulfil	  1	  or	  more	  of	  the	  following	  4	  inclusion	  criteria,	  which	  are	  all	  determined	  during	  
the	  conversation	  with	  the	  patient:	   
1.	  Ischemic	  heart	  disease,	  defined	  by	  any	  of	  the	  following	  criteria	  
	  
A.	  angina	  pectoris	  
	  
B.	  prior	  myocardial	  infarction	  
C.	  prior	  percutaneous	  coronary	  intervention	  	  

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D.	  prior	  coronary	  artery	  bypass	  graft	  	  
2.	  Peripheral	  arterial	  disease,	  defined	  by	  any	  of	  the	  following	  criteria	  	  
A.	  intermittent	  claudication	  	  
B.	  reduced	  peripheral	  arterial	  blood	  flow	  	  
C.	  prior	  vascular	  surgery	  due	  to	  peripheral	  arterial	  disease	  
3.	  Prior	  stroke	  
OR	  	  
4.	  any	  1	  of	  7	  risk	  criteria	  	  
A.	  age	  ≥70	  years;	  	  
B.	  congestive	  heart	  failure	  	  
C.	  prior	  transient	  ischemic	  attack;	  	  
D.	  diabetes	  and	  currently	  taking	  an	  oral	  hypoglycemic	  agent	  or	  
insulin	  	  
E.	  hypertension	  
G.	  preoperative	  serum	  creatinine	  >175	  µmol/L	  (>2.0	  mg/dl)	  	  
H.	  smoking	  within	  2	  years	  of	  surgery	  	  
Exclusion	  criteria	  
▪ 
History	  of	  peripheral	  arterial	  disease	  affecting	  both	  upper	  limbs	   
▪ 
Renal	  failure	  with	  eGRF<30ml/min/1.73m2 
▪ 
Cardiogenic	  shock	  or	  cardiac	  arrest	  during	  the	  current	  hospital	  admission 
▪ 
Reoperation	  after	  elective	  surgery	  carried	  out	  during	  the	  current	  hospital	  admission 
▪ 
Not	  capable	  of	  giving	  informed	  consent	  after	  oral	  and	  written	  information 
▪ 
Previously	  included	  in	  this	  trial 
	  
Interventions	  
Remote	  ischemic	  preconditioning:	  4	  cycles	  of	  forearm	  ischemia	  (200	  mmHg,	  5	  min)	  and	  
reperfusion	  (5	  min)	  immediately	  after	  induction	  of	  anesthesia.	  The	  procedure	  should	  be	  
performed	  on	  the	  limb	  opposite	  the	  one	  used	  for	  iv	  infusions.	  For	  patients	  with	  systolic	  blood	  
pressures	  >185	  mmHg,	  the	  cuff	  will	  be	  inflated	  to	  at	  least	  15	  mmHg	  above	  the	  patient’s	  
systolic	  blood	  pressure.	  	  
	  	  
Primary	  outcome	  

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▪ Number	  of	  patients	  with	  peak	  plasma	  cardiac	  troponin	  I	  (cTnI)	  ≥	  45ng/L	  (99th	  
percentile	  URL,	  10%	  CV	  at	  40ng/L)	  during	  surgery	  or	  during	  the	  first	  4	  days	  after	  
surgery	  (perioperative	  myocardial	  injury).	  If	  elevated	  at	  baseline1	  (preoperative)	  a	  
20%	  or	  greater	  increase	  of	  cTnI	  according	  to	  baseline	  is	  required	  for	  the	  diagnosis	  of	  a	  
perioperative	  myocardial	  injury	  [15].	  	  
 
Secondary	  outcome	  
▪ Peak	  plasma	  cTnI	  and	  total	  cTnI	  release	  (area	  under	  the	  curve)	  during	  surgery	  or	  
during	  the	  first	  4	  days	  after	  surgery 
▪ Endothelial	  dysfunction	  assessed	  by	  the	  non-­‐invasive	  EndoPat	  system	  (24	  hours	  and	  
72	  hours	  after	  surgery)	   
▪ Perioperative	  myocardial	  infarction	  (Universal	  definition	  of	  myocardial	  infarction,	  
published	  in	  2012	  [15])	   
▪ A	  typical	  rise	  or	  fall	  of	  cTnI	  with	  peak	  plasma	  cTnI	  ≥	  45ng/L	  (99th	  
percentile	  URL,	  10%	  CV	  at	  40	  ng/L)	  and	  with	  at	  least	  one	  of	  the	  
following: 
• 
Symptoms	  of	  ischemia	   
• 
New	  or	  presumed	  new	  significant	  ST-­‐segment-­‐T	  wave	  (ST-­‐T)	  
changes	  or	  new	  left	  bundle	  branch	  block	  (LBBB) 
• 
Development	  of	  pathologic	  Q	  waves	  in	  the	  ECG 
• 
Imaging	  evidence	  of	  new	  loss	  of	  viable	  myocardium	  or	  new	  
regional	  wall	  motion	  abnormality 
• 
Identification	  of	  an	  intracoronary	  thrombus	  by	  angiography	  or	  
autopsy 
▪ Cardiac	  death	  with	  symptoms	  suggestive	  of	  myocardial	  ischemia	  and	  
presumed	  new	  ischemic	  ECG	  changes	  or	  new	  LBBB,	  but	  death	  occurred	  
                                               
1 Defined as a preoperative cTnI	  ≥	  45ng/L	  (99th	  percentile	  URL,	  10%	  CV	  at	  40ng/L)	   

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before	  cardiac	  biomarkers	  were	  obtained,	  or	  before	  cardiac	  biomarker	  
values	  would	  be	  increased. 
▪ Development	  of	  new	  pathologic	  Q	  waves	  on	  an	  ECG	  or	  pathological	  
findings	  of	  a	  healing	  myocardial	  infarction	  if	  troponin	  levels	  were	  
obtained	  at	  times	  that	  could	  have	  missed	  the	  clinical	  event 
▪ Major	  adverse	  cardiovascular	  events	  (30	  days	  and	  one	  year	  after	  surgery)	   
▪ Nonfatal	  cardiac	  arrest,	  coronary	  revascularization	  procedure	  (PCI	  or	  
CABG),	  acute	  coronary	  syndrome,	  stroke,	  congestive	  heart	  failure,	  new	  
clinically	  important	  cardiac	  arrhythmia,	  peripheral	  arterial	  thrombosis,	  
rehospitalization	  for	  cardiovascular	  reasons.	   
▪ Length	  of	  postoperative	  hospital	  stay	   
▪ Length	  of	  intensive	  care	  unit	  stay 
▪ All-­‐cause	  mortality	  (30	  days	  and	  one	  year	  after	  surgery) 
▪ cardiovascular	  or	  non-­‐cardiovascular	  (a	  clearly	  documented	  non-­‐
cardiovascular	  cause) 
	  
Besides	  the	  inclusion	  and	  exclusion	  criteria,	  the	  following	  baseline	  data	  and	  
preoperative/postoperative	  risk	  factors	  will	  be	  collected	  from	  each	  patient	  (records):	  
- 
Age,	  sex	  
- 
co-­‐morbidities:	  chronic	  obstructive	  pulmonary	  disease,	  atrial	  fibrillation,	  active	  cancer	  	  
- 
medications	  (Paracetamol,	  NSAID,	  steroid,	  antibiotics,	  opioid,	  ASA,	  statin,	  brilique,	  
clopidogrel,	  anticoagulant	  medications,	  β-­‐blocker,	  calcium	  antagonist,	  diuretics,	  ACE-­‐I,	  
angiotensin	  antagonist,	  antiarytmic,	  nitrate,	  digoxin,	  antidiabetics	  incl.	  insulin)	  
preoperative	  and	  during	  hospitalization	  
- 
preoperative	  ASA	  score	  
- 
preoperative	  revised	  cardiac	  risk	  index	  score	  
- 
type	  and	  length	  of	  surgery	  
- 
type	  and	  length	  of	  anesthesia	  and	  type	  of	  analgesia	  	  

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- 
Intraoperative	  hypotension	  (and	  length)	  defined	  as	  a	  systolic	  blood	  pressure	  
<100mmHg	  	  
- 
Preoperative	  and	  postoperative	  plasma	  creatinine	  (incl.	  eGFR)	  and	  plasma	  hemoglobin	  
- 
blood	  transfusion	  
- 
Clinical	  events	  during	  hospitalization	  according	  to	  the	  Clavien	  Dindo	  classification	  [16,	  
17]	  e.g.	  pneumonia,	  sepsis,	  acute	  respiratory	  failure,	  acute	  kidney	  failure	  and	  lung	  
embolism.	  	  
	  
Participant	  timeline	  
Intervention:	  The	  remote	  ischemic	  preconditioning	  procedure	  is	  carried	  out	  immediately	  after	  
the	  induction	  of	  regional	  or	  general	  anesthesia.	  The	  blood	  pressure	  cuff	  is	  placed	  on	  the	  upper	  
limb.	  The	  cuff	  is	  inflated	  to	  200	  mmHg	  resulting	  in	  a	  total	  occlusion	  of	  the	  blood	  flow	  to	  the	  
limb.	  After	  5	  minutes	  of	  ischemia	  the	  cuff	  is	  deflated	  and	  the	  limb	  is	  reperfused	  for	  5	  minutes.	  
The	  procedure	  is	  carried	  out	  4	  times	  in	  a	  row.	  For	  patients	  with	  systolic	  blood	  pressures	  >185	  
mmHg,	  the	  cuff	  will	  be	  inflated	  to	  at	  least	  15	  mmHg	  above	  the	  patient’s	  systolic	  blood	  
pressure.	  	  
	  
The	  patients	  will	  have	  blood	  collected	  to	  assess	  cTnI	  (preoperative	  and	  in	  the	  morning	  at	  days	  
1,	  2,	  3	  and	  4	  after	  surgery).	  All	  patients	  with	  a	  cTnI	  assessment	  ≥	  45ng/L	  will	  have	  a	  minimum	  
of	  one	  ECG	  performed	  and	  a	  medical	  doctor	  will	  evaluate	  the	  patient	  for	  the	  presence	  of	  an	  
ischemic	  or	  non-­‐ischemic	  event	  that	  can	  explain	  the	  elevated	  cTnI	  (i.e.	  sepsis,	  pulmonary	  
embolism,	  cardioversion).	  The	  medical	  doctor	  will	  determine	  whether	  further	  action	  should	  
be	  taken.	  If	  necessary	  a	  cardiologist	  will	  be	  consulted.	  	  
	  
The	  investigator	  observes	  the	  patients	  during	  their	  stay	  in	  hospital	  and	  records	  the	  
occurrence	  of	  any	  primary	  or	  secondary	  outcomes.	  The	  investigator	  will	  undertake	  a	  short-­‐	  
and	  long-­‐term	  follow	  up	  (30	  days	  and	  1	  year	  after	  surgery)	  and	  record	  the	  occurrence	  of	  any	  
primary	  or	  secondary	  outcomes.	  The	  follow-­‐up	  will	  be	  carried	  out	  by	  reviewing	  the	  patients’	  
electronic	  hospital	  records	  and	  by	  a	  personal	  phone	  call.	  	  	  
	  
Sample	  size:	  A	  large	  international	  cohort	  study	  done	  by	  the	  VISION	  investigator	  group	  showed	  
that	  8.0%	  of	  patients	  undergoing	  non-­‐cardiac	  surgery	  suffered	  myocardial	  injury	  [4].	  In	  our	  

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study	  we	  include	  patients	  with	  a	  moderate	  –	  high	  risk	  of	  suffering	  myocardial	  injury,	  while	  the	  
VISION	  study	  included	  patients	  at	  all	  risk	  levels.	  We	  predict	  that	  15%	  of	  the	  patients	  in	  the	  
placebo	  group	  will	  suffer	  from	  myocardial	  injury	  and	  that	  the	  incidence	  of	  myocardial	  injury	  
will	  be	  reduced	  to	  7%	  in	  the	  intervention	  group.	  	  Type	  I	  error	  is	  set	  at	  5%	  and	  type	  II	  error	  is	  
set	  at	  20%.	  In	  total	  2	  x	  264	  patients	  need	  to	  be	  included	  based	  on	  this	  power	  calculation.	  We	  
will	  include	  patients	  until	  we	  have	  a	  total	  of	  2	  x	  270	  patients	  for	  evaluation.	  	  
We	  will	  do	  an	  interim	  analysis	  based	  on	  the	  primary	  outcome	  when	  a	  total	  of	  270	  (50%)	  
evaluable	  patients	  have	  been	  included	  in	  the	  study.	  We	  will	  use	  the	  O’Brien	  Fleming	  method	  
with	  a	  level	  of	  significance	  at	  p<0.05	  (first	  analysis)	  and	  p<0.048	  (second	  analysis).	  	  
	  
Recruitment	  
All	  patients	  hospitalized	  to	  undergo	  emergent	  or	  urgent	  major	  noncardiac	  surgery	  at	  
Department	  of	  Surgery	  and	  Department	  of	  Orthopedic	  Surgery,	  Køge	  Hospital,	  will	  as	  far	  as	  
possible	  be	  screened	  to	  determine	  whether	  they	  meet	  the	  inclusion	  and	  exclusion	  criteria.	  	  
	  
Allocation	  and	  blinding	  
An	  electronic	  generator	  will	  create	  the	  randomization	  list.	  	  
Each	  patient	  allocation	  is	  randomly	  selected	  using	  sealed	  opaque	  envelopes.	  It	  is	  not	  possible	  
to	  blind	  the	  staff	  or	  the	  patient.	  Assessor	  blinding	  will	  be	  performed	  and	  the	  unblinding	  will	  
take	  place	  after	  final	  data	  analysis	  has	  been	  made.	  An	  independent	  data	  analysis	  team	  
consisting	  of	  a	  statistician	  and	  an	  external	  medical	  scientist	  will	  perform	  interim	  analysis.	  	  
	  
Data	  collection	  methods	  
Blood	  samples	  
The	  blood	  will	  be	  collected	  from	  a	  larger	  vein	  (e.g.	  the	  cubital	  vein).	  	  
	  
Plasma	  cardiac	  troponin	  I	  
Plasma	  cTnI	  will	  be	  analyzed	  at	  the	  local	  department	  of	  biochemistry	  using	  Siemens	  
Healthcare	  Dimension	  Vista.	  	  
	  

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ECG	  
Trained	  healthcare	  professionals	  using	  standard	  ECG	  equipment	  will	  record	  the	  ECG.	  	  	  
	  
Withdrawal	  criteria	  and	  dropouts	  
The	  patient	  can	  at	  any	  time	  decide	  to	  discontinue	  his/hers	  participation	  in	  the	  trial.	  In	  case	  of	  
major	  serious	  per-­‐	  or	  postoperative	  complications/events,	  the	  investigator	  can	  decide	  to	  
withdraw	  the	  patient	  from	  the	  trial	  for	  the	  sake	  of	  the	  patient.	  	  
The	  number	  of	  dropouts	  and	  withdrawals	  will	  be	  reported	  in	  the	  final	  manuscript	  and	  
new	  participants	  will	  be	  included	  until	  2	  x	  270	  patients	  have	  completed	  the	  trial.	  In	  order	  to	  
complete	  the	  protocol	  the	  patient	  must	  have	  received	  the	  intervention/placebo	  and	  have	  a	  
minimum	  of	  two	  postoperative	  cTnI	  assessments	  done	  within	  the	  first	  4	  postoperative	  days	  
(the	  primary	  outcome).	  If	  the	  primary	  outcome	  is	  missing	  the	  short-­‐	  and	  long-­‐term	  follow-­‐up	  
will	  still	  be	  done	  and	  the	  patient	  will	  be	  included	  in	  the	  final	  analysis	  (intention-­‐to-­‐treat	  
analysis).	  The	  patient	  will	  dropout	  if	  reoperated	  within	  the	  first	  4	  postoperative	  days.	  
	  
Data	  
Data	  will	  be	  collected	  according	  to	  “Datatilsynet”	  and	  “Persondataloven”.	  The	  trial	  will	  be	  
approved	  by	  the	  regional	  ethics	  committee,	  and	  the	  trial	  will	  be	  registered	  on	  
www.clinicaltrials.gov	  before	  inclusion	  of	  the	  first	  patient.	  All	  collected	  data	  will	  be	  registered	  
in	  case	  report	  forms	  (CRFs)	  according	  to	  a	  list	  of	  source	  data.	  We	  will	  use	  paper	  CRFs,	  and	  
later	  all	  data	  will	  be	  entered	  into	  an	  electronic	  database	  for	  statistical	  analysis.	  Electronic	  data	  
will	  according	  to	  the	  approval	  by	  “Datatilsynet”	  be	  stored	  at	  an	  access	  limited	  drive.	  	  
 
Statistics 
All	  statistical	  analysis	  will	  be	  done	  with	  SPSS/PC+	  package	  (SSPS,	  Chicago,	  IL,	  USA)	  or	  the	  SAS	  
9.3	  package.	  Data	  will	  be	  analyzed	  using	  non-­‐parametric	  or	  parametric	  statistics	  depending	  
on	  the	  distribution	  of	  data.	  Differences	  are	  considered	  significant	  when	  p<	  0.048	  (final	  
analysis).	  We	  will	  do	  a	  per-­‐protocol	  analysis	  and	  an	  intention-­‐to-­‐treat	  analysis.	  	  
A	  medical	  doctor	  will	  evaluate	  all	  patients	  with	  a	  rise	  in	  cTnI	  ≥	  45ng/L	  in	  order	  to	  
determine	  whether	  the	  elevation	  in	  cTnI	  is	  due	  to	  an	  ischemic	  or	  non-­‐ischemic	  disease.	  In	  the	  

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final	  data	  analysis	  we	  will	  do	  an	  analysis,	  which	  includes	  all	  patients	  (ischemic	  and	  non-­‐
ischemic)	  and	  a	  second	  analysis,	  which	  will	  only	  include	  patients	  with	  an	  ischemic	  disease.	  	  
	  
Ethics	  and	  dissemination	  
Informed	  consent	  
Potential	  participants	  will	  be	  contacted	  personally	  prior	  to	  the	  operation	  by	  the	  
anaesthesiologist,	  who	  is	  in	  charge	  of	  the	  operation.	  The	  patients	  will	  receive	  oral	  and	  written	  
information	  regarding:	  
• 
The	  trial,	  and	  that	  we	  would	  like	  to	  ask	  he/she	  to	  participate	  in	  the	  trial. 
• 
That	  it	  is	  voluntary	  to	  participate,	  and	  the	  subject	  has	  the	  right	  to	  and	  is	  
encouraged	  to	  take	  time	  to	  consider	  carefully	  if	  he/she	  wants	  to	  
participate. 
• 
The	  subject	  has	  the	  right	  to	  bring	  a	  friend	  or	  family	  member	  to	  the	  oral	  
participant	  information 
• 
That	  the	  trial	  is	  approved	  by	  “Datatilsynet”	  and	  “den	  Videnskabsetiske	  
Komité” 
• 
That	  the	  subject	  at	  any	  time	  can	  cease	  to	  participate	  in	  the	  trial	  and	  if	  they	  
do	  it	  will	  not	  have	  any	  consequences	  for	  further	  treatment 
The	  meeting	  will	  take	  place	  in	  a	  calm	  and	  quiet	  environment.	  Since	  all	  included	  patients	  
require	  emergent	  or	  urgent	  surgery,	  it	  will	  not	  in	  all	  cases	  be	  possible	  to	  give	  the	  patient	  24	  
hours	  to	  consider	  participation.	  However,	  all	  patients	  will	  be	  given	  enough	  time	  to	  read	  the	  
written	  information,	  listen	  to	  the	  oral	  information	  and	  ask	  any	  questions.	  A	  similar	  setup	  is	  
required	  in	  a	  randomized	  clinical	  trial,	  which	  we	  are	  currently	  conducting.	  The	  study	  includes	  
patients	  in	  the	  acute	  phase	  of	  an	  acute	  myocardial	  infarction	  and	  was	  approved	  by	  “Den	  
Videnskabsetiske	  Komité,	  Region	  Hovedstaden”	  (Journal	  nummer:	  H-­‐3-­‐2010-­‐117,	  EudraCT	  
nummer:	  2010-­‐022400-­‐53).  
In	  the	  written	  information	  the	  amendment	  “Dine	  rettigheder	  som	  forsøgsperson	  i	  et	  
biomedicinsk	  forskningsprojekt”	  from	  “Den	  Centrale	  Videnskabsetiske	  Komités”	  pre-­‐printed	  

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folder	  will	  be	  included.	  The	  patient	  will	  be	  encouraged	  to	  read	  this	  before	  giving	  his	  informed	  
consent.	  It	  is	  central	  and	  important	  to	  investigate	  the	  remote	  ischemic	  preconditioning	  
intervention	  in	  a	  group	  of	  acute	  patients.	  Patients	  that	  require	  acute	  surgery	  have	  a	  much	  
higher	  risk	  than	  others	  for	  developing	  major	  cardiovascular	  complication.	  Therefore,	  it	  is	  of	  
particular	  importance	  to	  investigate	  if	  this	  non-­‐invasive	  intervention	  can	  improve	  the	  
treatment	  and	  outcome	  for	  this	  group	  of	  high-­‐risk	  patients.	  	  	  
	  
Safety	  
The	  use	  of	  a	  blood	  pressure	  cuff/tourniquet	  in	  remote	  ischemic	  preconditioning	  is	  considered	  
completely	  safe.	  There	  is	  a	  small	  possibility	  of	  having	  skin	  erythema	  in	  the	  area	  where	  the	  
inflated	  cuff	  was	  placed.	  In	  case	  of	  regional	  anesthesia	  the	  patient	  can	  experience	  numbness	  
and	  discomfort	  during	  the	  preconditioning	  procedure.	  The	  discomfort	  will	  be	  similar	  to	  a	  5-­‐
minute	  blood	  pressure	  assessment.	  An	  identical	  preconditioning	  protocol	  was	  used	  in	  prior	  
Danish	  clinical	  trials	  including	  patients	  with	  stroke	  [18]	  and	  acute	  myocardial	  infarction	  [19].	  
In	  one	  of	  the	  studies	  a	  questionnaire	  was	  designed	  to	  detect	  any	  discomfort	  created	  by	  the	  
preconditioning	  [18].	  No	  reports	  of	  significant	  discomfort	  were	  reported	  [18].	  The	  numbness	  
and	  skin	  erythema	  are	  both	  fully	  reversible	  and	  will	  rapidly	  wear	  off.	  There	  is	  a	  very	  small	  
possibility	  of	  getting	  a	  skin	  infection	  or	  haematoma	  after	  having	  the	  blood	  samples	  taken.	  We	  
will	  therefore	  use	  a	  standardised	  aseptic	  technique	  in	  trying	  to	  avoid	  this	  complication.	  	  
	  
Ethical	  considerations	  
We	  seek	  the	  approval	  from	  “den	  Videnskabsetiske	  Komité”	  and	  “Datatilsynet”.	  	  
The	  ischemic	  intervention	  is	  considered	  completely	  safe,	  and	  has	  been	  done	  in	  previous	  large	  
clinical	  trials	  [9,	  10].	  No	  serious	  adverse	  events	  have	  been	  reported	  [9,	  10].	  The	  patients	  will	  
gain	  a	  personal	  benefit	  by	  participating	  if	  this	  simple	  and	  low-­‐risk	  intervention	  is	  effective.	  
The	  risk	  of	  myocardial	  injury	  and	  postoperative	  cardiovascular	  morbidity	  could	  be	  markedly	  
reduced	  in	  the	  intervention	  group.	  The	  preconditioning	  procedure	  will	  not	  prolong	  the	  
anesthetic	  time	  or	  delay	  the	  onset	  of	  surgery.	  	  
Participation	  in	  the	  trial	  is	  voluntary.	  Patients	  will	  be	  given	  oral	  and	  written	  information	  
according	  to	  the	  Helsinki	  II	  declaration.	  The	  patients	  can,	  at	  any	  time,	  withdraw	  from	  the	  

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project,	  which	  will	  have	  no	  consequences	  for	  further	  treatment.	  The	  authorized	  healthcare	  
professionals	  involved	  in	  the	  project	  will	  demonstrate	  diligence	  in	  their	  duties.	  The	  
participating	  patients’	  integrity	  and	  autonomy	  will	  be	  respected.	  The	  trial	  will	  be	  carried	  out	  
at	  the	  Department	  of	  Surgery,	  Køge	  Hospital.	  
	  
Duration:	  
The	  trial	  is	  planned	  to	  initiate	  when	  all	  relevant	  approvals	  have	  been	  received	  (“Datatilsynet”,	  
“den	  Videnskabsetiske	  Komité”).	  We	  expect	  to	  include	  4-­‐6	  subjects	  each	  week.	  We	  expect	  to	  
start	  the	  trial	  in	  January	  2014	  and	  end	  it	  in	  January	  2016.	  
	  
Economy:	  
The	  patients	  will	  not	  receive	  any	  economic	  compensation	  for	  their	  participation.	  	  
The	  trial	  is	  initiated	  by:	  Sarah	  Ekeløf	  Busch	  (MD,	  PhD	  student),	  Ismail	  Gögenur	  (MD,	  
professor,	  DMSc)	  from	  Department	  of	  Surgery,	  Køge	  Hospital	  and	  Ole	  Mathiesen	  (MD,	  Ph.D.)	  
from	  Department	  of	  Anesthesiology,	  Køge	  Hospital.	  	  
	  
Funding	  
The	  trial	  has	  received	  funding	  from:	  
Toyota	  Fonden	  
	  
	  
100.000kr	  
Beckett-­‐Fonden	  
	  
	  
100.000kr	  
Snedkermester	  Sophus	  Jacobsens	  Fond	  
30.000kr	  
	  
The	  study	  was	  designed	  and	  will	  be	  conducted,	  analysed,	  interpreted,	  and	  reported	  
independently	  of	  the	  funding	  sources.	  	  
	  
	  
	  

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Publication	  
All	  results,	  positive	  as	  well	  as	  negative	  will	  be	  published	  in	  international	  journals	  with	  peer-­‐
review.	  Authors	  will	  all	  fulfill	  the	  ICMJE-­‐guidelines	  for	  authorship.	  	  
	  
Order	  of	  authors	  in	  all	  publications	  
1st	  author:	  Sarah	  Ekeløf	  Busch	  
2nd	  author:	  Ole	  Mathiesen	  
3rd	  author:	  Mikkel	  Holm	  Larsen	  
Last	  author:	  Ismail	  Gögenur	  
	  
More	  authors	  may	  be	  added	  if	  they	  according	  to	  the	  authors	  listed	  fulfill	  the	  ICMJE-­‐guidelines	  
for	  authorship.	  
	  
The	  possible	  significance	  of	  the	  trial 
Perioperative	  myocardial	  infarction	  and	  myocardial	  injury	  in	  noncardiac	  surgery	  is	  more	  
common	  than	  previously	  anticipated.	  At	  the	  moment	  no	  prevention	  or	  treatment	  is	  known	  
and	  universally	  accepted.	  Remote	  ischemic	  preconditioning	  is	  a	  safe,	  cheap	  and	  a	  fast	  
intervention,	  which	  can	  be	  easily	  implemented.	  If	  the	  intervention	  is	  effective	  we	  can	  
potentially	  reduce	  the	  perioperative	  and	  postoperative	  cardiovascular	  morbidity	  and	  
mortality.	  	  	  	  
	  
	  
	  
	  
	  
	  
	  
	  

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Referencer	  
	  
1.	  
Weiser,	  T.G.,	  et	  al.,	  An	  estimation	  of	  the	  global	  volume	  of	  surgery:	  a	  modelling	  strategy	  based	  on	  
available	  data.	  Lancet	  2008;372:139-­‐44.	  
2.	  
Devereaux,	  P.J.,	  et	  al.,	  Perioperative	  cardiac	  events	  in	  patients	  undergoing	  noncardiac	  surgery:	  a	  
review	  of	  the	  magnitude	  of	  the	  problem,	  the	  pathophysiology	  of	  the	  events	  and	  methods	  to	  
estimate	  and	  communicate	  risk.	  Cmaj	  2005;176:627-­‐34.	  
3.	  
Devereaux,	  P.J.,	  et	  al.,	  Effects	  of	  extended-­‐release	  metoprolol	  succinate	  in	  patients	  undergoing	  
non-­‐cardiac	  surgery	  (POISE	  trial):	  a	  randomised	  controlled	  trial.	  Lancet	  2008;371:1839-­‐47.	  
4.	  
Devereaux,	  P.J.,	  et	  al.,	  Association	  between	  postoperative	  troponin	  levels	  and	  30-­‐day	  mortality	  
among	  patients	  undergoing	  noncardiac	  surgery.	  Jama	  2012;307:2295-­‐304.	  
5.	  
Landesberg,	  G.,	  et	  al.,	  Perioperative	  myocardial	  infarction.	  Circulation	  2009;119:2936-­‐44.	  
6.	  
Kharbanda,	  R.K.,	  T.T.	  Nielsen,	  and	  A.N.	  Redington,	  Translation	  of	  remote	  ischaemic	  
preconditioning	  into	  clinical	  practice.	  Lancet	  2009;374:1557-­‐65.	  
7.	  
Hausenloy,	  D.J.	  and	  D.M.	  Yellon,	  Remote	  ischaemic	  preconditioning:	  underlying	  mechanisms	  and	  
clinical	  application.	  Cardiovasc	  Res	  2008;79:377-­‐86.	  
8.	  
Tapuria,	  N.,	  et	  al.,	  Remote	  ischemic	  preconditioning:	  a	  novel	  protective	  method	  from	  ischemia	  
reperfusion	  injury-­‐-­‐a	  review.	  J	  Surg	  Res	  2008;150:304-­‐30.	  
9.	  
Thielmann,	  M.,	  et	  al.,	  Cardioprotective	  and	  prognostic	  effects	  of	  remote	  ischaemic	  
preconditioning	  in	  patients	  undergoing	  coronary	  artery	  bypass	  surgery:	  a	  single-­‐centre	  
randomised,	  double-­‐blind,	  controlled	  trial.	  Lancet	  2013;382:597-­‐604.	  
10.	  
Botker,	  H.E.,	  et	  al.,	  Remote	  ischaemic	  conditioning	  before	  hospital	  admission,	  as	  a	  complement	  to	  
angioplasty,	  and	  effect	  on	  myocardial	  salvage	  in	  patients	  with	  acute	  myocardial	  infarction:	  a	  
randomised	  trial.	  Lancet,	  2010;375:727-­‐34.	  
11.	  
Li,	  C.,	  et	  al.,	  Limb	  remote	  ischemic	  preconditioning	  for	  intestinal	  and	  pulmonary	  protection	  
during	  elective	  open	  infrarenal	  abdominal	  aortic	  aneurysm	  repair:	  a	  randomized	  controlled	  trial.	  
Anesthesiology	  2013;118:842-­‐52.	  
12.	  
Li,	  C.,	  et	  al.,	  Limb	  Remote	  Ischemic	  Preconditioning	  Attenuates	  Lung	  Injury	  after	  Pulmonary	  
Resection	  under	  Propofol-­‐Remifentanil	  Anesthesia:	  A	  Randomized	  Controlled	  Study.	  
Anesthesiology	  2014.	  
13.	  
Hu,	  S.,	  et	  al.,	  Effects	  of	  remote	  ischemic	  preconditioning	  on	  biochemical	  markers	  and	  neurologic	  
outcomes	  in	  patients	  undergoing	  elective	  cervical	  decompression	  surgery:	  a	  prospective	  
randomized	  controlled	  trial.	  J	  Neurosurg	  Anesthesiol	  2010;22:46-­‐52.	  
14.	  
Huang,	  J.,	  et	  al.,	  Effect	  of	  remote	  ischaemic	  preconditioning	  on	  renal	  protection	  in	  patients	  
undergoing	  laparoscopic	  partial	  nephrectomy:	  a	  'blinded'	  randomised	  controlled	  trial.	  BJU	  Int	  	  
2013;112:74-­‐80.	  
15.	  
Thygesen,	  K.,	  et	  al.,	  Third	  universal	  definition	  of	  myocardial	  infarction.	  J	  Am	  Coll	  Cardiol	  
2012;60:1581-­‐98.	  
16.	  
A	  clinical	  trial	  comparing	  primary	  coronary	  angioplasty	  with	  tissue	  plasminogen	  activator	  for	  
acute	  myocardial	  infarction.	  The	  Global	  Use	  of	  Strategies	  to	  Open	  Occluded	  Coronary	  Arteries	  in	  
Acute	  Coronary	  Syndromes	  (GUSTO	  IIb)	  Angioplasty	  Substudy	  Investigators.	  N	  Engl	  J	  Med	  
1997;336:1621-­‐8.	  
17.	  
Dindo,	  D.,	  N.	  Demartines,	  and	  P.A.	  Clavien,	  Classification	  of	  surgical	  complications:	  a	  new	  
proposal	  with	  evaluation	  in	  a	  cohort	  of	  6336	  patients	  and	  results	  of	  a	  survey.	  Ann	  Surg	  
2004;240:205-­‐13.	  
18.	  
Hougaard,	  K.D.,	  et	  al.,	  Remote	  ischemic	  perconditioning	  as	  an	  adjunct	  therapy	  to	  thrombolysis	  in	  
patients	  with	  acute	  ischemic	  stroke:	  a	  randomized	  trial.	  Stroke	  2014;45:159-­‐67.	  
19.	  
Sloth,	  A.D.,	  et	  al.,	  Improved	  long-­‐term	  clinical	  outcomes	  in	  patients	  with	  ST-­‐elevation	  myocardial	  
infarction	  undergoing	  remote	  ischaemic	  conditioning	  as	  an	  adjunct	  to	  primary	  percutaneous	  
coronary	  intervention.	  Eur	  Heart	  J	  2014;35:168-­‐75.	  

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Prevention of myocardial injury by remote ischemic 
preconditioning in emergent or urgent non-cardiac 
surgery: a randomized clinical trial 
The PIXIE study 
 
Contact information 
Primary investigator: Sarah Ekeløf Busch, MD, Ph.D.-student, Department of Surgery, 
Zealand University Hospital, Lykkebækvej 1, 4600 Køge.  
Sponsor, investigator: Ismail Gögenur, Professor, MD, DMSc, Department of Surgery, Zealand 
University Hospital, Lykkebækvej 1, 4600 Køge.  
Investigator: Ole Mathiesen, MD, Ph.D., Department of Anesthesiology, Zealand University 
Hospital, Lykkebækvej 1, 4600 Køge.  
Investigator: Morten Homilius, MD, Department of Orthopedic Surgery, Holstebro Hospital, 
Lægårdvej 12, 7500 Holstebro 
Investigator: Christian Meyhoff, MD, Ph.D., Department of Anesthesiology, Herlev Hospital, 
Herlev Ringvej 75, 2730 Herlev 
Investigator: Ossian Gundel, Stud. Med., Department of Surgery, Zealand University Hospital, 
Lykkebækvej 1, 4600 Køge. 
 
Applicant: Sarah Ekeløf Busch, MD, Ph.D.-student, Department of Surgery, Zealand University 
Hospital, Lykkebækvej 1, 4600 Køge. 
Tlf: +45 22 71 28 74 
E-mail: sarahekeloef@gmail.com  
 
Signature/Date: 
 / 29.12.2016 
 
Applicant, Sarah Ekeløf Busch 

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Introduction  
Worldwide, more than 200 million patients have major non-cardiac surgery annually and a 
significant proportion of these patients suffer major cardiovascular complications (e.g. 
nonfatal myocardial infarction, cardiac arrest, vascular death) within 30 days of their surgery 
[1, 2]. Perioperative myocardial infarction is the most common cardiovascular complication 
and recent clinical studies have shown that even minor myocardial injury in relation to non-
cardiac surgery is associated with 30-day mortality [3, 4].  
The pathogenesis of perioperative myocardial infarction is complex and not fully 
elucidated. Surgery is associated with trauma, anesthesia and analgesia, bleeding and anemia, 
pain, hypothermia and fasting [2]. These factors initiate systemic inflammation, 
hypercoagulability, increased levels of catecholamines and cortisol and a state of hypoxia [2]. 
The drastic systemic changes can lead to a myocardial hypoxic supply-demand mismatch 
where rupture of vulnerable coronary plaques might represent an additional mechanism [2, 
5]. At the moment, no effective prevention or treatment of perioperative myocardial 
infarction in non-cardiac surgery is accepted.  
The aim of this study is to determine whether an intervention with remote ischemic 
preconditioning can reduce myocardial injury in emergent or urgent non-cardiac surgery.  
 
Remote ischemic preconditioning 
Remote ischemic preconditioning is a procedure, which protects remote tissues and 
organs e.g. against ischemia-reperfusion injury [6]. Cycles of forearm or leg ischemia and 
reperfusion by the inflation of a blood-pressure cuff for brief periods are the preferred 
method [7]. The procedure is simple, safe and with no clear side effects [6].  
The mechanism of remote ischemic preconditioning is not fully understood. However, it 
seems that the procedure triggers complex endogenous pathways including a humoral 
pathway, a neural pathway and a systemic anti-inflammatory pathway [7, 8].  
In clinical trials covering acute cardiology and cardiac surgery, remote ischemic 
preconditioning has effectively reduced myocardial injury, postoperative cardiovascular 
complications and cardiac mortality [9, 10]. Recently, the effect of remote ischemic 
preconditioning on attenuating ischemia-reperfusion injury has been investigated in non-
cardiac surgery [11-14]. The organ specific ischemia-reperfusion injury, systemic oxidative 

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stress and inflammatory response were attenuated due to the intervention [11-14]. Whether 
remote ischemic preconditioning protects against myocardial injury in the perioperative 
period of non-cardiac surgery have until now not been investigated.  
 
Aim 
- 
To determine whether remote ischemic preconditioning can reduce markers of 
myocardial injury in emergent or urgent non-cardiac surgery.  
 
Methods 
Trial design 
Randomized clinical trial    
 
Study setting 
Department of Surgery, Zealand University Hospital, Denmark 
Department of Orthopedic Surgery, Zealand University Hospital, Denmark 
Department of Anesthesiology, Zealand University Hospital, Denmark 
Department of Orthopedic Surgery, Holstebro Hospital, Denmark 
Department of Clinical Biochemistry, Holstebro Hospital, Denmark  
Department of Anesthesiology, Herlev Hospital, Denmark 
Department of Orthopedic Surgery, Herlev Hospital, Denmark 
 
Eligibility criteria 
Inclusion criteria 
▪ 
patients ≥ 45 years undergoing in-hospital major non-cardiac surgery  
▪ 
major orthopedic surgery  
• 
hip surgery  
▪ 
undergoing emergent or urgent surgery (i.e. emergent or urgent visitation to the 
Department of Orthopedic Surgery) 
▪ 
fulfil 1 or more of the following 4 inclusion criteria, which are all determined during 
the conversation with the patient:  
1. Ischemic heart disease, defined by any of the following criteria 
 
A. angina pectoris 

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B. prior myocardial infarction 
C. prior percutaneous coronary intervention  
D. prior coronary artery bypass graft  
2. Peripheral arterial disease, defined by any of the following criteria  
A. intermittent claudication  
B. reduced peripheral arterial blood flow  
C. prior vascular surgery due to peripheral arterial disease 
3. Prior stroke 
OR  
4. any 1 of 7 risk criteria  
A. age ≥70 years;  
B. congestive heart failure  
C. prior transient ischemic attack;  
D. diabetes and currently taking an oral hypoglycemic agent or 
insulin  
E. hypertension 
G. preoperative serum creatinine >175 µmol/L (>2.0 mg/dl)  
H. smoking within 2 years of surgery  
Exclusion criteria 
▪ History of peripheral arterial disease affecting both upper limbs  
▪ Renal failure with eGRF<30ml/min/1.73m2 
▪ Cardiogenic shock or cardiac arrest during the current hospital admission 
▪ Reoperation after elective surgery carried out during the current hospital admission 
▪ Other conditions that prevent the performance of remote ischemic preconditioning.  
▪ Not capable of giving informed consent after oral and written information 
▪ Previously included in this trial 
 
Interventions 
Remote ischemic preconditioning: 4 cycles of forearm ischemia (200 mmHg, 5 min) and 
reperfusion (5 min) immediately after induction of anesthesia. The procedure should be 
performed on the limb opposite the one used for iv infusions. For patients with systolic blood 

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pressures >185 mmHg, the cuff will be inflated to at least 15 mmHg above the patient’s 
systolic blood pressure.  
 
Primary outcome 
▪ 
Number of patients with peak plasma cardiac troponin I (cTnI) ≥ 45ng/L (99th 
percentile URL, 10% CV at 40ng/L) during surgery or during the first 4 days after 
surgery (perioperative myocardial injury). If elevated at baseline1 (preoperative) a 
20% or greater increase of cTnI according to baseline is required for the diagnosis of a 
perioperative myocardial injury [15].  
o Holstebro Hospital: Number of patients with peak plasma cardiac troponin I 
(cTnI) > 24 ng/L (99th percentile URL, 14% CV) 
 
Secondary outcome 
▪ 
Peak plasma cTnI and total cTnI release (area under the curve) during surgery or 
during the first 4 days after surgery 
▪ 
Plasma N-terminal Pro-Brain Natriuretic Peptide (NT-proBNP) before surgery and at 
day 1 after surgery. 
▪ 
Endothelial dysfunction assessed by the non-invasive EndoPat system (24 hours after 
surgery, only done at Zealand University Hospital)  
▪ 
Biomarkers of coagulation (only patients included at Holstebro Hospital) 
▪ 
Perioperative myocardial infarction (Universal definition of myocardial infarction, 
published in 2012 [15])  
▪ 
A typical rise or fall of cTnI with peak plasma cTnI ≥ 45ng/L (99th 
percentile URL, 10% CV at 40 ng/L) (Holstebro cTnI > 24 ng/L), and with 
at least one of the following: 
• 
Symptoms of ischemia  
                                                 
1 Defined as a preoperative cTnI ≥ 45ng/L (99th percentile URL, 10% CV at 40ng/L)  

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• 
New or presumed new significant ST-segment-T wave (ST-T) 
changes or new left bundle branch block (LBBB) 
• 
Development of pathologic Q waves in the ECG 
• 
Imaging evidence of new loss of viable myocardium or new 
regional wall motion abnormality 
• 
Identification of an intracoronary thrombus by angiography or 
autopsy 
▪ 
Cardiac death with symptoms suggestive of myocardial ischemia and 
presumed new ischemic ECG changes or new LBBB, but death occurred 
before cardiac biomarkers were obtained, or before cardiac biomarker 
values would be increased. 
▪ 
Development of new pathologic Q waves on an ECG or pathological 
findings of a healing myocardial infarction if troponin levels were 
obtained at times that could have missed the clinical event 
▪ 
Major adverse cardiovascular events (30 days, one year and five years after surgery)  
▪ 
Nonfatal cardiac arrest, coronary revascularization procedure (PCI or 
CABG), acute coronary syndrome, stroke, congestive heart failure, new 
clinically important cardiac arrhythmia, peripheral arterial thrombosis, 
rehospitalization for cardiovascular reasons.  
▪ 
Length of postoperative hospital stay  
▪ 
Length of intensive care unit stay 
▪ 
All-cause mortality (30 days, one year and five years after surgery) 
▪ 
cardiovascular or non-cardiovascular (a clearly documented non-
cardiovascular cause) 
 
Besides the inclusion and exclusion criteria, the following baseline data and 
preoperative/postoperative risk factors will be collected from each patient (records): 
- 
Age, sex 

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- 
co-morbidities: chronic obstructive pulmonary disease, atrial fibrillation, active cancer, 
autoimmune diseases  
- 
medications (Paracetamol, NSAID, steroid, antibiotics, opioid, ASA, statin, brilique, 
clopidogrel, anticoagulant medications, β-blocker, calcium antagonist, diuretics, ACE-I, 
angiotensin antagonist, antiarytmic, nitrate, digoxin, antidiabetics incl. insulin) 
preoperative and during hospitalization 
- 
preoperative ASA score 
- 
preoperative revised cardiac risk index score 
- 
type and length of surgery 
- 
type and length of anesthesia and type of analgesia  
- 
Intraoperative hypotension (and length) defined as a systolic blood pressure 
<100mmHg  
- 
Preoperative and postoperative plasma creatinine (incl. eGFR) and plasma hemoglobin 
- 
blood transfusion 
- 
Clinical events during hospitalization according to the Clavien Dindo classification [16, 
17] e.g. pneumonia, sepsis, acute respiratory failure, acute kidney failure and lung 
embolism.  
 
The one and five year follow-up on all included patients will be performed with information 
(procedure- and diagnosis codes) registered in the Danish National Patients Register 
(Landspatientsregisteret). Likewise, information on death and cause of death will be deducted 
from “Dødsårsagsregisteret”.  
 
Participant timeline 
Intervention: The remote ischemic preconditioning procedure is carried out immediately after 
the induction of regional or general anesthesia. The blood pressure cuff is placed on the upper 
limb. The cuff is inflated to 200 mmHg resulting in a total occlusion of the blood flow to the 
limb. After 5 minutes of ischemia the cuff is deflated and the limb is reperfused for 5 minutes. 
The procedure is carried out 4 times in a row. For patients with systolic blood pressures >185 
mmHg, the cuff will be inflated to at least 15 mmHg above the patient’s systolic blood 
pressure. The first round should be completed before the initiation of surgery.  
 

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The patients will have blood collected to assess cTnI (preoperative and in the morning at days 
1, 2, 3 and 4 after surgery) and NT proBNP (preoperative and in the morning at day 1 after 
surgery). All patients with a cTnI assessment ≥ 45ng/L will have a minimum of one ECG 
performed and a medical doctor will evaluate the patient for the presence of an ischemic or 
non-ischemic event that can explain the elevated cTnI (i.e. sepsis, pulmonary embolism, 
cardioversion). The medical doctor will determine whether further action should be taken. If 
necessary, a cardiologist will be consulted. Patient included at Holstebro Hospital will have 
blood collected for assessment of coagulation biomarkers. The blood samples are taken 
preoperatively, 2 hours after incision and in the morning at day 1 and 2 after surgery (4 times 
6 ml blood).  
 
The investigator observes the patients during their stay in hospital and records the 
occurrence of any primary or secondary outcomes. The investigator will undertake a short- 
and long-term follow up (30 days, one and five years after surgery) and record the occurrence 
of any primary or secondary outcomes. The follow-up will be carried out by reviewing the 
patients’ electronic hospital records, by a personal phone call and with data from the Danish 
National Patient Register and “Dødsårsagsregisteret”.   
 
Sample size: A large international cohort study done by the VISION investigator group showed 
that 8.0% of patients undergoing non-cardiac surgery suffered myocardial injury [4]. In our 
study we include patients with a moderate – high risk of suffering myocardial injury, while the 
VISION study included patients at all risk levels. We predict that 15% of the patients in the 
placebo group will suffer from myocardial injury and that the incidence of myocardial injury 
will be reduced to 7% in the intervention group.  Type I error is set at 5% and type II error is 
set at 20%. In total 2 x 264 patients need to be included based on this power calculation. We 
will include patients until we have a total of 2 x 270 patients for evaluation.  
 
Biobank 
We will establish a biobank at Department of Clinical Biochemistry, Holstebro Hospital, 
containing the drawn plasma for analysis of biomarkers of coagulation and NT-pro-BNP. Only 
patients included at Holstebro Hospital will have samples drawn for the biobank. In total, a 

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maximum of 30 ml blood will be drawn from each participating patient (24mL for the 
coagulation biomarkers and 6 ml for NT-pro-BNP). The blood samples will be centrifuged and 
only plasma samples will be stored (-80OC). Moreover, a biobank will be established at the 
Department of Surgery, Zealand University Hospital. Every 4-6 month plasma samples for NT-
pro-BNP analysis will transferred securely from the biobank at Holstebro Hospital to the 
biobank at Zealand University Hospital. All plasma samples will be retained until all analyzes 
are made and for no more than 10 years. The purpose of the biobank is to make sure that all 
assessments are made with the same equipment in the lab, hereby reducing variations due to 
technical variations in the equipment used. All requirements regarding establishment and 
maintenance of the biobank will be met. 
 
Recruitment 
All patients hospitalized to undergo emergent or urgent major noncardiac surgery at 
Department of Orthopedic Surgery, Zealand University Hospital, Department of Orthopedic 
Surgery, Holstebro Hospital, and Department of Orthopedic Surgery, Herlev Hospital, will as 
far as possible be screened to determine whether they meet the inclusion and exclusion 
criteria.  
 
Allocation and blinding 
An electronic generator will create the randomization list.  
Each patient allocation is randomly selected using sealed opaque envelopes. It is not possible 
to blind the staff or the patient. Assessor blinding will be performed and the unblinding will 
take place after final data analysis has been made. An independent data analysis team 
consisting of a statistician and an external medical scientist will perform interim analysis.  
 
Data collection methods 
Blood samples 
The blood will be collected from a larger vein (e.g. the cubital vein).  
 
 
 

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Plasma cardiac troponin I 
Plasma cTnI will be analyzed at the local department of biochemistry using Siemens 
Healthcare Dimension Vista (Zealand University Hospital, Herlev Hospital), Abbott high-
sensitive troponin I (Holstebro Hospital).  
 
Plasma NT proBNP 
Plasma NT proBNP will be analyzed in a standardized manner at the Department of 
Biochemistry. Samples from Holstebro Hospital will be analyzed as one batch in Region 
Zealand when all patients have been included. Until then the plasma samples will be stored in 
a biobank at Department of Biochemistry (Holstebro Hospital) and Department of Surgery 
(Zealand University Hospital).  
 
Coagulation biomarkers 
The blood is centrifuged and the remaining plasma is stored in the bio-bank at -80OC until 
further analysis.  
 
ECG 
Trained healthcare professionals using standard ECG equipment will record the ECG.   
 
Withdrawal criteria and dropouts 
The patient can at any time decide to discontinue his/hers participation in the trial. In case of 
major serious per- or postoperative complications/events, the investigator can decide to 
withdraw the patient from the trial for the sake of the patient.  
The number of dropouts and withdrawals will be reported in the final manuscript and 
new participants will be included until 2 x 270 patients have completed the trial. In order to 
complete the protocol the patient must have received the intervention/placebo and have a 
minimum of two postoperative cTnI assessments done within the first 4 postoperative days 
(the primary outcome). If the primary outcome is missing the short- and long-term follow-up 
will still be done and the patient will be included in the final analysis (intention-to-treat 
analysis). The patient will dropout if reoperated within the first 4 postoperative days. 
 
 

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Data 
Data will be collected according to “Datatilsynet” and “Persondataloven”. The trial will be 
approved by the regional ethics committee, and the trial will be registered on 
www.clinicaltrials.gov before inclusion of the first patient. All collected data will be registered 
in case report forms (CRFs) according to a list of source data. We will use paper CRFs, and 
later all data will be entered into an electronic database for statistical analysis. Electronic data 
will according to the approval by “Datatilsynet” be stored at an access limited drive.  
 
Statistics 
All statistical analysis will be done with SPSS/PC+ package (SSPS, Chicago, IL, USA) or the SAS 
9.3 package. Data will be analyzed using non-parametric or parametric statistics depending 
on the distribution of data. Differences are considered significant when p< 0.05. We will do a 
per-protocol analysis and an intention-to-treat analysis.  
A medical doctor will evaluate all patients with a rise in cTnI ≥ 45ng/L in order to 
determine whether the elevation in cTnI is due to an ischemic or non-ischemic disease. In the 
final data analysis, we will do an analysis, which includes all patients (ischemic and non-
ischemic) and a second analysis, which will only include patients with an ischemic disease.  
 
Ethics and dissemination 
Informed consent 
Potential participants will be contacted personally prior to the operation by the 
anaesthesiologist or surgeon, who is in charge of the operation. The patients will receive oral 
and written information regarding: 
• The trial, and that we would like to ask he/she to participate in the trial. 
• That it is voluntary to participate, and the subject has the right to and is 
encouraged to take time to consider carefully if he/she wants to 
participate. 

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• The subject has the right to bring a friend or family member to the oral 
participant information 
• That the trial is approved by “Datatilsynet” and “den Videnskabsetiske 
Komité” 
• That the subject at any time can cease to participate in the trial and if they 
do it will not have any consequences for further treatment 
• Patient at Holstebro Hospital: We have established a biobank at 
Department of Clinical Biochemistry (Holstebro Hospital) and Department 
of Surgery (Zealand University Hospital). Their plasma for assessment of 
coagulation markers and NT-pro-BNP will be stored until further analysis.  
The meeting will take place in a calm and quiet environment. Since all included patients 
require emergent or urgent surgery, it will not in all cases be possible to give the patient 24 
hours to consider participation. However, all patients will be given enough time to read the 
written information, listen to the oral information and ask any questions. A similar setup is 
required in a randomized clinical trial, which we are currently conducting. The study includes 
patients in the acute phase of an acute myocardial infarction and was approved by “Den 
Videnskabsetiske Komité, Region Hovedstaden” (Journal nummer: H-3-2010-117, EudraCT 
nummer: 2010-022400-53).  
 
In the written information the amendment “Dine rettigheder som forsøgsperson i et 
biomedicinsk forskningsprojekt” from “Den Centrale Videnskabsetiske Komités” pre-printed 
folder will be included. The patient will be encouraged to read this before giving his informed 
consent. It is central and important to investigate the remote ischemic preconditioning 
intervention in a group of acute patients. Patients that require acute surgery have a much 
higher risk than others for developing major cardiovascular complication. Therefore, it is of 
particular importance to investigate if this non-invasive intervention can improve the 
treatment and outcome for this group of high-risk patients.   
 
 

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Safety 
The use of a blood pressure cuff/tourniquet in remote ischemic preconditioning is considered 
completely safe. There is a small possibility of having skin erythema in the area where the 
inflated cuff was placed. In case of regional anesthesia, the patient can experience numbness 
and discomfort during the preconditioning procedure. The discomfort will be similar to a 5-
minute blood pressure assessment. An identical preconditioning protocol was used in prior 
Danish clinical trials including patients with stroke [18] and acute myocardial infarction [19]. 
In one of the studies a questionnaire was designed to detect any discomfort created by the 
preconditioning [18]. No reports of significant discomfort were reported [18]. The numbness 
and skin erythema are both fully reversible and will rapidly wear off. There is a very small 
possibility of getting a skin infection or haematoma after having the blood samples taken. We 
will therefore use a standardised aseptic technique in trying to avoid this complication.  
 
Ethical considerations 
We seek the approval from “den Videnskabsetiske Komité” and “Datatilsynet”.  
The ischemic intervention is considered completely safe, and has been done in previous large 
clinical trials [9, 10]. No serious adverse events have been reported [9, 10]. The patients will 
gain a personal benefit by participating if this simple and low-risk intervention is effective. 
The risk of myocardial injury and postoperative cardiovascular morbidity could be markedly 
reduced in the intervention group. The preconditioning procedure will not prolong the 
anesthetic time or delay the onset of surgery.  
Participation in the trial is voluntary. Patients will be given oral and written information 
according to the Helsinki II declaration. The patients can, at any time, withdraw from the 
project, which will have no consequences for further treatment. The authorized healthcare 
professionals involved in the project will demonstrate diligence in their duties. The 
participating patients’ integrity and autonomy will be respected. The trial will be carried out 
at the Department of Surgery, Zealand University Hospital. 
 
 

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Duration: 
The trial is planned to initiate when all relevant approvals have been received (“Datatilsynet”, 
“den Videnskabsetiske Komité”). We expect to include 4-6 subjects each week. We expect to 
start the trial in January 2014 and end it in January 2018. 
 
Economy: 
The patients will not receive any economic compensation for their participation.  
The trial is initiated by: Sarah Ekeløf Busch (MD, PhD student), Ismail Gögenur (MD, 
professor, DMSc) from Department of Surgery, Zealand University Hospital and Ole Mathiesen 
(MD, Ph.D.) from Department of Anesthesiology, Zealand University Hospital.  
 
Funding 
The trial has received funding from: 
Toyota Fonden 
 
 
100.000kr 
Beckett-Fonden 
 
 
100.000kr 
Snedkermester Sophus Jacobsens Fond 
30.000kr 
 
The study was designed and will be conducted, analyzed, interpreted, and reported 
independently of the funding sources.  
 
 
 
 
 
 
 

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Budget 
EXPENSES 
SPECIFICATION 
UNIT 
PER UNIT/KR 
TOTAL/KR 
EQUIPMENT 
Endopat 2000 (Itamar 
Medical Ltd., Caesarea, 
Israel) 
1 
161,250 
161,250 
 
Finger probes 
2160 
1,980kr/box with 12 
units (180 boxes) 
356,400 
ANALYSIS 
Troponin I 
2700 
30 
81,000 
 
Pro-BNP 
1080 
180 
194,400 
WAGES 
Scholarship 
 
 
120,000 
SUM 
 
 
 
913,050 
OVERHEAD (3.1%) 
 
 
 
28,304.55 
TOTAL 
 
 
 
941,354.55 
 
Publication 
All results, positive as well as negative will be published in international journals with peer-
review. Authors will all fulfill the ICMJE-guidelines for authorship.  
Order of authors in all publications 
1st author: Sarah Ekeløf Busch 
2nd author: Ole Mathiesen 
Last author: Ismail Gögenur 
More authors may be added if they according to the authors listed fulfill the ICMJE-guidelines 
for authorship. 
The possible significance of the trial 
Perioperative myocardial infarction and myocardial injury in noncardiac surgery is more 
common than previously anticipated. At the moment no prevention or treatment is known 
and universally accepted. Remote ischemic preconditioning is a safe, cheap and a fast 
intervention, which can be easily implemented. If the intervention is effective, we can 
potentially reduce the perioperative and postoperative cardiovascular morbidity and 
mortality.    
 

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Referencer 
 
1. 
Weiser, T.G., et al., An estimation of the global volume of surgery: a modelling strategy based on 
available data. Lancet 2008;372:139-44. 
2. 
Devereaux, P.J., et al., Perioperative cardiac events in patients undergoing noncardiac surgery: a 
review of the magnitude of the problem, the pathophysiology of the events and methods to 
estimate and communicate risk. Cmaj 2005;176:627-34. 
3. 
Devereaux, P.J., et al., Effects of extended-release metoprolol succinate in patients undergoing 
non-cardiac surgery (POISE trial): a randomised controlled trial. Lancet 2008;371:1839-47. 
4. 
Devereaux, P.J., et al., Association between postoperative troponin levels and 30-day mortality 
among patients undergoing noncardiac surgery. Jama 2012;307:2295-304. 
5. 
Landesberg, G., et al., Perioperative myocardial infarction. Circulation 2009;119:2936-44. 
6. 
Kharbanda, R.K., T.T. Nielsen, and A.N. Redington, Translation of remote ischaemic 
preconditioning into clinical practice. Lancet 2009;374:1557-65. 
7. 
Hausenloy, D.J. and D.M. Yellon, Remote ischaemic preconditioning: underlying mechanisms and 
clinical application. Cardiovasc Res 2008;79:377-86. 
8. 
Tapuria, N., et al., Remote ischemic preconditioning: a novel protective method from ischemia 
reperfusion injury--a review. J Surg Res 2008;150:304-30. 
9. 
Thielmann, M., et al., Cardioprotective and prognostic effects of remote ischaemic 
preconditioning in patients undergoing coronary artery bypass surgery: a single-centre 
randomised, double-blind, controlled trial. Lancet 2013;382:597-604. 
10. 
Botker, H.E., et al., Remote ischaemic conditioning before hospital admission, as a complement to 
angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a 
randomised trial. Lancet, 2010;375:727-34. 
11. 
Li, C., et al., Limb remote ischemic preconditioning for intestinal and pulmonary protection 
during elective open infrarenal abdominal aortic aneurysm repair: a randomized controlled trial. 
Anesthesiology 2013;118:842-52. 
12. 
Li, C., et al., Limb Remote Ischemic Preconditioning Attenuates Lung Injury after Pulmonary 
Resection under Propofol-Remifentanil Anesthesia: A Randomized Controlled Study. 
Anesthesiology 2014. 
13. 
Hu, S., et al., Effects of remote ischemic preconditioning on biochemical markers and neurologic 
outcomes in patients undergoing elective cervical decompression surgery: a prospective 
randomized controlled trial. J Neurosurg Anesthesiol 2010;22:46-52. 
14. 
Huang, J., et al., Effect of remote ischaemic preconditioning on renal protection in patients 
undergoing laparoscopic partial nephrectomy: a 'blinded' randomised controlled trial. BJU Int  
2013;112:74-80. 
15. 
Thygesen, K., et al., Third universal definition of myocardial infarction. J Am Coll Cardiol 
2012;60:1581-98. 
16. 
A clinical trial comparing primary coronary angioplasty with tissue plasminogen activator for 
acute myocardial infarction. The Global Use of Strategies to Open Occluded Coronary Arteries in 
Acute Coronary Syndromes (GUSTO IIb) Angioplasty Substudy Investigators. N Engl J Med 
1997;336:1621-8. 
17. 
Dindo, D., N. Demartines, and P.A. Clavien, Classification of surgical complications: a new 
proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg 
2004;240:205-13. 
18. 
Hougaard, K.D., et al., Remote ischemic perconditioning as an adjunct therapy to thrombolysis in 
patients with acute ischemic stroke: a randomized trial. Stroke 2014;45:159-67. 
19. 
Sloth, A.D., et al., Improved long-term clinical outcomes in patients with ST-elevation myocardial 
infarction undergoing remote ischaemic conditioning as an adjunct to primary percutaneous 
coronary intervention. Eur Heart J 2014;35:168-75. 

Summary of Changes 
Protocol 
Amendment, date 
Details 
1. Amendment, 
04.02.2015  
Adding the assessment of N-terminal pro-Brain Natriuretic 
Peptide (Zealand University Hospital trial site) 
2. Amendment, 
04.06.2015 
Adding a trial site (Regional Hospital West Jutland) 
At the new trial site, high-sensitive troponin I was assessed.  
Myocardial injury was defined as high-sensitive troponin I > 24 
ng/L (99th percentile URL, 14% CV)  
3. Amendment, 
13.08.2015 
Adding a trial site (Herlev Hospital) 
Excluding patients undergoing major abdominal surgery 
Adding an assessment of endothelial function and coagulation 
markers 
4. Amendment, 
25.01.2016 
Adding the assessment of N-terminal pro-Brain Natriuretic 
Peptide (Regional Hospital West Jutland trial site) 
5. Amendment, 
29.12.2016 
Adding the exclusion criteria: “Other conditions that prevent the 
performance of remote ischemic preconditioning” 
Expanding the clinical follow-up until 5 years after surgery 
 

Original version 
 
1 
 
Prevention of myocardial injury by remote ischemic preconditioning in 
emergent or urgent non-cardiac surgery: a randomized clinical trial 
The PIXIE study 
Statistical analysis plan for the primary publication 
 
Definitions of the population 
Intention-to-treat: randomized in the trial and a minimum of two postoperative troponin 
assessments within four days of surgery (an evaluable primary outcome) 
Per-protocol: randomized in the trial, received the allocated treatment, and a minimum of two 
postoperative troponin assessments within four days of surgery 
We will do a per-protocol analysis and an intention-to-treat analysis.  
 
The primary analysis 
Comparing the incidence of myocardial injury (No. (%)) in the intervention group versus the 
control group. We plan to use a chi-square test. The difference is considered to be significant if 
the two-sided p-value < 0.05. 
 
Secondary analyses  
All analyses compare the difference between the intervention group and the control group. 
Continuous outcomes will be analyzed with non-parametric (Wilcoxon two-sample test) or 
parametric statistics (unpaired t-test) depending on data distribution and equality of 
variances. If necessary data will be logarithm transformed. Continuous outcomes will be 
expressed as mean (95% confidence interval) or median (inter-quartile range). Categorical 
outcomes will be analyzed with a chi-square test or Fischer’s exact test (an expected count < 
five in one cell) and the result will be expressed as no. (%). Differences are considered 
significant if the two-sided p-value < 0.05. 
Secondary comparisons include 
- 
Peak plasma troponin I within four days of surgery 
- 
Total troponin I release (area under the curve) during the first four days after surgery 
- 
Perioperative myocardial infarction 

Original version 
 
2 
 
- 
Major adverse cardiovascular events (30 days) 
- 
Length of postoperative hospital stay  
- 
Length of intensive care unit stay  
- 
All-cause mortality (30 days) 
 
Sample size 
We predict that 15% of the patients in the placebo group will suffer from myocardial injury 
and that the incidence of myocardial injury will be reduced to 7% in the intervention group.  
Type I error is set at 5% and type II error is set at 20%. In total 2 x 264 patients need to be 
included based on this power calculation. We will include patients until we have a total of 2 x 
270 patients for evaluation (per-protocol).  
We will do an interim analysis based on the primary outcome when a total of 270 (50%) 
evaluable patients have been included in the study. We will use the O’Brien Fleming method 
with a level of significance at p<0.005 (first analysis) and p<0.048 (second analysis).  
 

Final version 
 
1 
 
Prevention of myocardial injury by remote ischemic preconditioning in 
emergent or urgent non-cardiac surgery: a randomized clinical trial 
The PIXIE study 
Statistical analysis plan for the primary publication 
 
Definitions of the population 
Intention-to-treat: randomized in the trial and a minimum of two postoperative troponin 
assessments within four days of surgery (an evaluable primary outcome) 
Per-protocol: randomized in the trial, received the allocated treatment, and a minimum of two 
postoperative troponin assessments within four days of surgery 
We will do a per-protocol analysis and an intention-to-treat analysis.  
 
The primary analysis 
Comparing the incidence of myocardial injury (No. (%)) in the intervention group versus the 
control group. We plan to use a chi-square test. The difference is considered to be significant if 
the two-sided p-value < 0.05. 
 
Secondary analyses  
All analyses compare the difference between the intervention group and the control group. 
Continuous outcomes will be analyzed with non-parametric (Wilcoxon two-sample test) or 
parametric statistics (unpaired t-test) depending on data distribution and equality of 
variances. If necessary data will be logarithm transformed. Continuous outcomes will be 
expressed as mean (95% confidence interval) or median (inter-quartile range). Categorical 
outcomes will be analyzed with a chi-square test or Fischer’s exact test (an expected count < 
five in one cell) and the result will be expressed as no. (%). Differences are considered 
significant if the two-sided p-value < 0.05. 
Secondary comparisons include 
- 
Peak plasma troponin I within four days of surgery (stratified on cardiac troponin I 
and high-sensitive troponin I) 

Final version 
 
2 
 
- 
Total troponin I release (area under the curve) during the first four days after surgery 
(stratified on cardiac troponin I and high-sensitive troponin I) 
- 
Perioperative myocardial infarction 
- 
Major adverse cardiovascular events (30 days) 
- 
Length of postoperative hospital stay  
- 
Intensive care unit admission 
- 
All-cause mortality (30 days) 
 
To analyze the association between the intervention and the primary outcome, myocardial 
injury, we will do a univariate and multivariate logistic regression. The results will be 
expressed as odds ratios (95% confidence interval).   
The decision to include a variable in the multivariate logistic regression will be based on 
clinical hypotheses. Trial site effect will be analyzed. A maximum of one variable per 10 
primary events will be included.  
 
Sample size 
We predict that 15% of the patients in the placebo group will suffer from myocardial injury 
and that the incidence of myocardial injury will be reduced to 7% in the intervention group.  
Type I error is set at 5% and type II error is set at 20%. In total 2 x 264 patients need to be 
included based on this power calculation. We will include patients until we have a total of 2 x 
270 patients for evaluation (per-protocol).  
 

Summary of Changes 
Statistical Analysis Plan 
 
Amendment, date 
Details 
1. Amendment, 
04.06.2015 
Peak plasma troponin I and total troponin I release (area under 
the curve): The analyses will be stratified on cardiac troponin I 
and high-sensitive troponin I 
2. Amendment, 
25.01.2016 
We decided not to perform the interim analysis.  
No data analysis had been performed and the investigators 
were blinded.  
3. Amendment, 
01.09.2017 
The addition of a univariate and multivariate logistic regression 
to analyze the association between the intervention and the 
primary outcome. The analyses were added before the study 
inclusion was terminated. No data analysis had been performed 
and the investigators were blinded.