Sore Throat Secondary to Endotracheal Intubation: the Role of Neutrophil Activation
Phase: NA
Conditions: Patients Requiring Endotracheal Intubation
Sponsor: Washington University School of Medicine

RESEARCH PROTOCOL 
 
 
 
STUDY TITLE 
Sore throat secondary to endotracheal intubation: the role of neutrophil activation 
 
 
 
 
 
 
 
Carlos A. Puyo, MD, FCCP 
Assistant Professor  
Washington University School of Medicine 
Department of Anesthesiology 
660 S. Euclid  
Campus Box 8054 
St. Louis MO 63110 
Voice: 314-747-0259 
 
E-mail: puyoc@wustl.edu 
 
Andrew Gelman, PhD 
Assistant Professor of Surgery 
Washington University School of Medicine 
Department of Surgery 
660 S. Euclid  
Campus Box 8054 
St. Louis MO 63110 
Voice: 314-362-8382 
Email- gelmana@wustl.edu 
 
 
 
Version Date:  Original: February 21.2013 
Amendment: June 27, 2013 
Amendment: August 12, 2013 
Amendment: September 30, 2013 
Amendment: December 20, 2013 
Amendment: January 24, 2014 
Amendment: March 13, 2014 
 

 
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Revised March 13, 2014January 11, 2016 
1.  SYNOPSIS 
 
Study Title 
Sore throat secondary to endotracheal intubation: the role of neutrophil 
activation 
Objective 
The study is aimed at identifying neutrophil surface changes that occur 
during tracheal injury secondary to endotracheal intubation and to 
correlate them with the development of sore throat. 
Study Period 
Planned enrollment duration: 1 year 
 
Planned study duration: Subjects will participate from time of consent 
prior to surgery through approximately 120 minutes following 
extubation.   
 
Number of Patients 
60 evaluable patients and 25 evaluable healthy controls 
 
Study Treatment  
Patients will undergo placement of an endotracheal tube (ETT) as 
standard of care. Tracheal lavage will be conducted using 5 mL of 
sterile saline solution by a push/suction technique. Specimen samples 
obtained by wall suction will be collected at 2 time points following 
intubation.  
 
 Blood samples will be obtained at 2 time points, simultaneous with the 
collection of the tracheal specimens.  
 
Healthy volunteers will have a blood draw. 
Study Design 
This study is prospective. 
Inclusion and 
Exclusion Criteria 
Inclusion: 
1. Ages 18 to 65 
2. American Society of Anesthesiologists Physical Status 
Classification (ASA) I and II as determined by the study 
physician as this is a subjective measure. 
3. Required to have endotracheal intubation 
4. Scheduled for short admission (<23 hrs), or ambulatory 
orthopedic surgery (ankle, shoulder, or extremity surgery); 
general surgery (exploratory laparotomy, cholecystectomy, 
ventral or inguinal hernia repair); obstetric/gynecological 
surgery(total abdominal hysterectomy, bilateral salpingo-
oophorectomy); urologic surgery (nephrectomy, 
prostatectomy), lumbar spine surgery( discectomy, one or 
two level lumbar fusion) procedures 

 
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Revised March 13, 2014January 11, 2016 
5. Provide informed consent. 
Exclusion: 
1. Active pulmonary disease within 5 days prior to enrollment 
2. On immunosuppressive medications 
3. Previous tracheal surgery 
4. On azithromycin 
5. Diabetes 
6. Pregnancy 
7. Smoking history of less than 6 weeks prior to surgery 
8. Surgery or intubation within 5 days of surgery 
9. Planned surgical procedure involving neck or thoracic regions 
10. Active pulmonary disease 
11. Autoimmune disease (HIV, AIDS, Rheumatoid Arthritis 
12. Hepatitis B or C, or active Hepatitis A 
13. Cancer 
Healthy Controls: Inclusion: 
1.  Ages 18 to 65 
2.  Provide informed consent. 
Measurements 
Lavage Processing 
Tracheal lavage fluid specimens will be placed on ice and transported 
immediately to our laboratory. A sample of 0.25cc or less will be 
removed for cell count and differential count.  We will do total cell 
counts using tryptan blue to check viability of cells. Remaining whole 
fluid will be centrifuged at 1500 rpm for 15 minutes at 4° C and 
supernatants frozen in aliquots.  
Neutrophil assessment and purification 
Neutrophils will be counted in the tracheal lavage and peripheral blood 
with a HEMAVET analyzer (Drew Scientific). Neutrophils will be 
purified by immunomagnetic bead-mediated negative selection with 
biotin-labeled antibodies (e-biosciences) specific for CD11b, CD11c, 
CD14,  CD16 CD62L, CD54, CD32, CD88, and CD66b. Flow 
cytometry will be used for assessment of neutrophil surface receptor 
presence. 
Reactive Oxygen Species (R0S) determination 
Neutrophil production of ROS will be assessed with the fluoro-probe 
dihydrorhodamine. 
Interleukin Assays 
Accumulation of interleukin 1 (IL-1), IL-4, IL-6, IL-8, IL-10, IL-12, IL-
17, IL-23p19, (transformin growth factor 1 (TGF β-1) and tumor 
necrosis factor alpha (TNF-α) in culture will follow stimulation with 
formylated peptides by immunosorbent assay (ELISA) for human 

 
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Revised March 13, 2014January 11, 2016 
cytokines (R&D Systems, Minneapolis, MN). 
Toll-like Receptors (TLR) 
Determination of TLR-2, TLR-4 and TLR-9 and Mitochondrial DNA 
will be done by PCR. 
Neutrophil extracellular traps (NETs) formation 
Neutrophil cells will be stained and photographic documentation of the 
NET presence will be documented. 
Statistical 
Methodology 
The primary aim of this study is to describe the inflammatory changes 
that result in a neutrophil surface receptor expression unique to the 
interaction between the trachea and ETT resulting in sore throat. Blood 
samples will be obtained and neutrophil cells (PMN) will be isolated for 
analysis at the same time intervals as the tracheal lavage samples. 
Individual indicators of inflammation (PMN, cytokines, clusters of 
differentiation –CD, TLR’s as described above) will be compared to 
baseline values of these variables measured at time zero. We will 
compare neutrophil (PMN) changes between patients who developed 
sore throat and those who do not by using the Wilcoxon rank sum test, 
since we expect the data to be non-normally distributed. With a sample 
size of 60 patients, we will have at least 80% power to detect large 
differences (Cohen’s D≥O.8) between the sore throat and non-sore 
throat groups with an alpha level of 0.05 and assuming that half of the 
participants report a sore throat following intubation. As our pilot study 
is exploratory, we did not inflate our sample size to account for multiple 
testing. We will confirm any identified associations found in our pilot 
study with a future larger study. Subjects who undergo intubation 
attempts x 2 or greater will be considered a subset population and not 
part of the 60 evaluable patients. Assuming that lack of sore throat 
relates to anti-inflammation, measuring a biomarker such as IL-10, may 
provide information on the regulatory function of this cytokine. We 
have observed that IL-10 at a concentration of 200 pg/ml generated by 
PMNs inhibited reactive oxygen species burst from alveolar 
macrophages challenged by LPS. Power analysis was conducted for two 
tailed T-test using the following assumptions:  a standard deviation of 
100 pg/ml. A difference of 75 pg/ml between the two groups will be 
considered a significant difference. We will need approximately 30 
patients per group for an alpha 0.05 and Beta of 0.2.  
 

 
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Revised March 13, 2014January 11, 2016 
2.  STUDY PROTOCOL  
 
2.1 Background and Significance 
Background and preliminary Studies: 
 
Endotracheal intubation results in a high incidence of sore throat. Placement of an endotracheal 
tube (ETT) is essential for the administration of general anesthesia. Approximately 60 thousand 
surgical cases are performed every day in the United States that require general anesthesia. The ETT 
has been implicated in local tracheal injury, development of postoperative sore throat (POST)1,2,3,4,5,6, 
tracheitis and pneumonia7. POST is ranked high by patients along with pain and nausea as an 
undesirable outcome following general surgery1,2,3,4.  Although sore throat tends to be more evident 
in the first 24 hours, its presence has a negative influence on patient satisfaction post-surgery2,4. The 
incidence of POST has been reported around 50% in several studies following general endotracheal 
anesthesia and appears to affect a broad range of patients1,2,3,4. Some studies have shown a greater 
incidence of sore throat in young female patients who received succinylcholine, and underwent 
gynecological procedures; other studies have not shown a significant gender difference4. Although 
the exact pathophysiology of POST has not been elucidated, we recently showed that various arms of 
the innate immune system are activated following ETT placement5,6. Notably, we observed the 
expression of inflammatory mediators, complement C5a activation and neutrophil infiltration in the 
mucosal tissues and the trachea in the absence of bacterial infection.  However, we did observe 
mucosal surface injury. As it has been clearly shown that neutrophil infiltration and activation can be 
driven in response to sterile cell death8, our preliminary data suggest that the link between ETT 
placement and neutrophil accumulation is through the injury to mucosal cells.  
 
PMN infiltration found significant in our previous studies of tracheal inflammation. In a pilot 
swine study5 we documented a reproducible pattern of inflammation in the peritracheal area in which 
all markers measured displayed a positive increase over a period of time (6 hours). A similar pattern 
was documented in our human study6 in which we found that despite intubating under controled 
conditions by an experienced practitioner, tracheal injury was still demonstrated (Figure 1). In both 
studies (swine and human), we measured cellular concentration (polymorphonuclear cells) and a 
group of proteins (cytokines) that indicate cellular responses to injury. In both groups, a significant 
neutrophilic reaction was documented, hence the reason for the study. 
 
Significance of PMN phenotype in local tracheitis induced by endotracheal intubation. If our 
hypothesis is correct we predict that patients with sore throat will have a different PMN pro-
inflammatory activation phenotype compared to patients with no sore throat, and we anticipate that 
the neutrophils will have a regulatory phenotype (IL-10 expression).  Determination of PMN 
phenotypes will be essential in future research planning as sore throat may represent a group of 
patients more prone to developing active airway inflammation and bacterial seeding in different 
segments of the lower airways. PMN activation is a desirable effect to clean up acute inflammatory 
products but unopposed inflammatory activity may be more detrimental to local tissue homeostasis 
preventing the resolution of inflammation.   

 
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Revised March 13, 2014January 11, 2016 
 
Endotracheal intubation relates to Ventilator-associated pneumonia. The exact etiology between 
tracheal injury and lower airway inflammation is quite unclear.  The prevailing view is that 
intubation-induced inflammation is caused by bacterial pathogens introduced by the endotracheal 
tube.  However, our previous studies have suggested little evidence of bacterial pathogens in the 
upper airway, with ample evidence of upper tracheal epithelial injury, neutrophil recruitment and 
inflammatory cytokine release during short term intubation. Thus, we hypothesize that intubation-
induced inflammation is predominantly a sterile tracheal injury response that may compromise 
homeostatic barriers important in preventing subsequent infection.  Interestingly, recent studies have 
suggested the existence of different neutrophil subtypes with pro- and anti-inflammatory 
(immunosuppressive) functions12.  Some patients may avoid the consequences of upper airway injury 
by recruiting neutrophil populations with immunosuppressive factors that promote tissue regeneration 
and the resolution of inflammation.  
Importantly, several reports have suggested there is a relationship between endotracheal intubation 
and subsequent development of ventilator-associated pneumonia (VAP) with clear implications on 
overall lung function and patient morbidity. In particular, studies of ventilator-associated 
tracheobronchitis (VAT), indicate that 10- 32% of intubated patients diagnosed with VAT developed 
VAP33. The use of non-invasive positive pressure ventilation in COPD, respiratory insufficiency, 
immuno-compromised and congestive heart failure patients have shown to decrease the incidence of 
VAP dramatically, supporting the notion that the endotracheal tube is a major culprit in respiratory 
inflammation and infection34.   
The exact pathophysiology of endotracheal tube-associated pneumonia has not been elucidated 
although the endotracheal tube has been viewed as a major factor in development of VAP. A variety 
of endotracheal tube modifications (different cuff design, antibiotic coating, and addition of suction 
channels) have been designed to limit 
bacterial colonization and VAP have 
shown only small improvement in outcome 
for our patients 35 suggesting the need to 
better understand how  endotracheal tubes 
promote tracheal inflammation to better 
prevent distal airway diseases such as VAT 
and VAP. 
 
Sore 
throat 
and 
PMN 
elevation 
correlated in intubated patients. We 
conducted a human study of tracheal 
intubation and asked patients to described 
the presence or absence of sore throat after 
surgery, and found that the PMN elevation  
(figure 1) was slightly different between  
symptomatic 
and 
non-symptomatic 
subjects, albeit the population number was 
 
 
Figure 1. Polymorphonuclear cells (PMNs) as a percentage of total 
cells in human trachea lavages obtained from 11subjects who 
responded to post-operative sore throat questionnaire. Median time to 
end of surgery was 3 hours.  
 

 
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small. The initial markers of inflammation detected, such as PMN and protein, increased steadily 
(p<0.01) over time (average 3.2 hours) in human subjects. Neutrophils and cytokines are important 
components of an organized inflammatory reaction and since several of the cytokines documented 
may have their origin in the PMN cells, we decided to investigate PMN activation. There are studies 
evaluating the airway response to injury or infection, but to our knowledge, none have been related to 
upper tracheal inflammation and in particular the activation of PMN cells in the development of sore 
throat. 
Cytokines produced by PMNs participate in modulation of inflammation and anti-
inflammation. Cytokines are proteins produced by a variety of cells including neutrophils9. We 
measured several cytokines (TNF-α, IL-1β, IL-6, and IL-8) in the human tracheal specimens 
obtained. Our findings included a progressive elevation of all cytokines measured. TNF-α was not 
found initially but after the first hour it became significant (p<0.01). Importantly, some cytokines can 
delay neutrophil apoptosis, which can lead to substantial increases in mucosal inflammation10. One 
particular cytokine TNF-α has pro or anti-apoptotic actions in neutrophils depending on whether Nfk-
β is activated, a key transcriptional regulator that inhibits cell death11. Besides controlling survival, 
cytokines released by neutrophils can promote chemotaxis (IL-8), stimulate revascularization 
(VEGF), promote activation of other myeloid cells (GM-CSF, IL-6, IL-1b) and resolve inflammation 
(IL-10)12. Our observation regarding PMN derived cytokines and their participation in an 
inflammatory response is consistent with findings described in several studies9,10,11,12   . 
 
PMN activation occurs in response to bacterial and non-bacterial stimulants. Neutrophils are 
mobilized from the bone marrow and travel to a site of injury or infection following specific 
chemoattractant gradients released by injured tissue. Several chemoattractants such as interleukin 8 
(IL-8), leukotrienes, prostaglandins, complement 5a (C5a), platelet activating factor (PAF) and 
formylated 
peptides 
(fMLP) 
are 
known 
to 
control 
local 
neutrophil 
accumulation13. 
Lipopolysaccharide (LPS) is present in gram-negative bacteria and may promote the release of 
inflammatory cytokines such as tumor necrosis factor (TNF-α) and IL-1β. Neutrophils can be 
activated not only due to bacterial infiltration but also may be activated under sterile conditions14. 
Danger associated molecular patterns (DAMPS) released by injured cells (e.g. mitochondrial DNA, 
ATP, Heat shock proteins) following ischemia-reperfusion injury, trauma or chemical injury are 
recognized by pattern recognition receptors (PRR) which in turn generate innate immune responses15. 
One well characterized class of PRRs, are Toll-like receptors (TLRs). TLR2 and TLR4, for example 
recognize the DAMP heat shock protein 60 while TLR9 recognizes mitochondrial DNA. 
Interestingly, DAMPS mimic the effects of microbial pathogen-associated molecular patterns 
(PAMPS)15,16 such as LPS and can drive neutrophilic infiltration without the presence of microbial 
antigens15. Sterile inflammation results when tissue injury occurs in the absence of infectious 
organisms and presents a clinical picture characterized by local redness, swelling, heat and pain.  
 
Surface expression of encoded proteins is present in PMN cell during injury. Continuous 
exposure to an antigen will result in neutrophil surface expression –Clusters of Differentiation (CD)-, 
mediated by β2-integrins which are expressed only in neutrophil cells17. Cellular adhesion to plasma 
proteins such as intracellular adhesion molecule-1 (ICAM-1)18 (CD54) is mediated by integrins in 

 
Page 8 of 21 
 
 
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agreement by the recipient that no unpublished information contained herein will be published or disclosed without prior written 
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Revised March 13, 2014January 11, 2016 
response to inflammatory cytokines. Clusters of differentiation are used for identification of 
neutrophils, to determine migration, phagocytosis and activation. Some of the CDs expressed in the 
neutrophil surface include those related to neutrophil adhesion19 (CD11b, CD11c, CD16 CD62L, and 
CD54); Immunoglobulins (CD32) and anaphylatoxins (CD88, CD66b)20. The effects of granulocyte-
macrophage colony stimulating factor (GM-CSF), are well described regarding encoding of CDs in 
response to bacteria and DAMPs21; with significant enhancement of the pro-inflammatory activity of 
PMNs, and is consistent with the concept of a significant biosynthetic capacity for PMN cells. 
 
Neutrophils produce large quantities of reactive oxygen species. Although reactive oxygen 
species (ROS) are generated by neutrophils to control infection, this activity may also alter local 
healthy tissue. Hydrogen peroxide and superoxide are generated during phagocytosis of particles or 
elevated concentration of chemoattractants22. A sign of neutrophil activation is an acute increase in 
oxygen consumption (respiratory burst) that is mediated by a membrane-bound complex, 
nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase23. NADPH mediates 
formation of superoxide anion after dismutation to hydrogen peroxide, which in turn is involved in 
promoting formation of secondary reactive products, such as singlet oxygen, hypochlorous acid and 
hydroxyl radicals. The end point of this process is neutrophil degranulation with subsequent enzyme 
degradation of host and microbial tissues. 
 
Regulatory PMN cells are important for host protection against injury. PMN cells have 
demonstrated dual properties in regulating a specific response to sterile and non-sterile injury by 
using different mechanisms to induce inflammation or anti-inflammation (immunosuppression)24. 
During infectious injury mediated by gram positive, gram negative bacteria, fungus, PMN may 
influence activation of T cells which produce interferon γ (IFN- γ), and in turn IFN- γ acts as a potent 
activator of PMN locally25; or can involve Toll –like Receptors (TLRs) such as TLR-2, TLR-4 and 
TLR-926.  We are interested in studying the response of PMNs to injury secondary to the ETT, by 
analyzing several indicators of PMN activation regarding inflammation and anti-inflammation.    
 
Several systemic diseases and certain antibiotics may alter PMN responsiveness to injury. A 
variety of systemic diseases such as diabetes, rheumatoid arthritis, acute lung injury and several 
others may affect local PMN responses to injury by altering inflammation, apoptosis and proper anti-
inflammation. In the case of diabetes with microangiopathy surface receptor CD11b was elevated and 
CD62L was decreased27. The impact of macrolide antibiotics and in particular azithromycin in the 
improved outcome of respiratory diseases such as acute lung injury (ALI), community-acquired 
pneumonia (CAP) and ventilator associated pneumonia (VAP) appear to be mediated by its anti-
inflammatory properties. In particular azithromycin may affect PMN activation, PMN survival, 
oxidative burst, and attenuation of nuclear factor-κβ28, therefore modulating the inflammatory 
response. It is not known the exact effect of these systemic factors in the local expression of 
neutrophils at the tracheal level, therefore our interest to compare local and systemic PMN activation 
in the healthy subject. 
 

 
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A variety of biomaterials activate PMN in blood. The endotracheal tubes contain a biomaterial 
known as di-(2-ethyl-hexyl)-phthalate or DEHP as it is commonly known. Studies in human and rat 
blood have shown its ability to increased CD-11b expression in PMN cells29. The effects on PMN of 
neonates also include inhibition of chemotaxis, stimulation of oxidative metabolism, and upregulation 
of NADPH-oxidase-130. DEHP is the only plasticizer approved for human devices by the U.S. Food 
and Drug Administration. Studies of materials involved either human blood samples, or in vitro, or 
rat models, none have in vivo local activation of PMN cells. 
 
Significance of intubation related sore-throat in airway inflammation VAT/VAP: 
Placement of an endotracheal tube results in tracheal injury. We hypothesize that intubation-related 
sore throat is associated with neutrophil activation in the airway.  To the best of our knowledge the 
association of neutrophil activation in the subglottic area and intubation-related sore throat has not 
been studied.  We contend this is a relevant clinical phenomenon, which has implications beyond the 
larynx and pharynx because studies have shown that there is a link between upper (endotracheal 
intubation) and lower respiratory inflammation or infection (VAP)36. Although not all patients 
develop sore throat following intubation they do have elevated neutrophils in the subglottic area, 
suggesting the possibility of the existence of different neutrophils phenotypes regulating 
inflammation in the trachea. Therefore, to provide better insight into the pathophysiology of upper 
airway inflammation and progression to VAT and VAP we propose to analyze PMN-mediated 
inflammation in the trachea through characterizing neutrophil phenotype and function in our patient 
population.  
Neutrophils have a fundamental role in inflammation as effectors cells, capable of responding to 
sterile tissue injury. Expression of surface receptors on the neutrophils under certain conditions may 
result in an activated phenotype that will enhance their ability to migrate, produce more reactive 
oxygen species and delay apoptosis. Unique PMN receptors –cluster of differentiation CD- had been 
found in blood of patients with respiratory diseases (asthma, COPD) and had been correlated with 
severity of clinical manifestations. Neutrophil activity has been extensively investigated during 
systemic inflammation; however, the characterization of neutrophil phenotype and function in local 
sites of inflammation is less well defined. Placement of an endotracheal tube (ETT) is performed for 
a variety of medical conditions including short term general anesthesia. We are interested in 
analyzing the neutrophil phenotype in vivo while the ETT is in direct contact with the tracheal tissue 
in an attempt to correlate them with the occurrence of sore throat. A better understanding of 
neutrophil trafficking and CD expression in the upper airway may offer new and more precise 
therapeutic targets to prevent local and distant disease. 
The glutathione-S-transferase mu1 (GMSTM1) null genotype is fairly common in the general 
population (40-43%) and in all ethnicities (Montero R. et al. 2007. Hum Biol). The presence of 
GMSTM1 is associated with increased risk for cancer (Zhong S, et al. 1993. Carcinogenesis) and 
inflammatory diseases of the airway and lungs (Alexis NE, et al. 2009. J Allergy Clin Immunol; Wu 
W, et al. 2012. Free Radic Biol Med). Studies have shown that reactive oxygen species (ROS) are 
abnormally generated in GMSTM1-null phenotype (Yang Y, et al. 2009. Hypertension). Since 
neutrophil activity is closely link to ROS activity and we have demonstrated in our studies of human 
tracheal lavages that ROS is significantly elevated in those with tracheal injury, we would want to 

 
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Revised March 13, 2014January 11, 2016 
analyze the presence of GMSTM1 in all the specimens collected and in the prospective human 
donors.    
 
Effect of ATG16L1 allelic variation on neutrophil longevity.  Autophagy 16-like 1 (ATG16L1) is 
an essential part of a protein complex responsible for initiating and carrying out autophagy. 
Autophagy is an essential intracellular process necessary for intracellular degradation and involves 
autophagosome and lysosome formation. Most people carry non-mutant, or “hypermorphic,” alleles 
for ATG16L1 allowing for the proper degradation of unneeded intracellular components. However, 
some carry mutant, or “hypomorphic,” alleles that disrupt normal autophagic processes. This 
disruption in proper autophagy leads to improper and excessive inflammatory responses by the innate 
immune system. Patients both heterozygous and homozygous for these hypomorphic alleles have a 
markedly increased risk of developing inflammatory bowel diseases, particularly Crohn’s disease. 
It has been shown that the presence of the hypomorphic ATG16L1 alleles also contribute to the 
development of ventilator associated pneumonia and other postoperative inflammatory 
complications32. However, the effect these alleles have on postoperative responses by the most 
prominent inflammatory immune cell the neutrophil, have yet to be elucidated. By isolation and 
subsequent sequencing of these alleles in neutrophils from patients with and without postoperative 
inflammatory complications, we can further understand how this gene plays a role in thoracic 
immune response. 
Mitochondrial DNA released during cellular injury or trauma has been implicated in sterile 
inflammation. Injured cells release a variety of molecules capable of eliciting an innate immune 
response to non-bacterial products. The presence of mitochondrial DNA (mtDNA) released during 
cell injury has been implicated in development of non-bacterial or sterile inflammation by activation 
of human neutrophils leading to increased migration, activation and degranulation mediated by Ca2+ 
and mitogen-activated protein kinases (MAPK). Studies of trauma patients with evidence of systemic 
inflammation indicate that DAMPs are present in the serum of affected patients and in particular 
mtDNA has been detected at levels several times higher than patients without trauma. Less is known 
of local cellular injury and in particular in the tracheal area during active injury such as endotracheal 
tube presence. The mechanisms involved in PMN activation resulting in tracheal and airway injury 
have not been elucidated, hence our interest in studying mtDNA locally with the goal to determine its 
impact on PMN activation and thus design therapies directed to specific anti-inflammatory targets 
using anti-mitocondrial DNA37.  
Neutrophil are essential cells of the innate immune system and have multiple defensive functions 
intended to protect tissue homeostasis. As part of the extracellular neutrophil activity a release of a 
substance call neutrophil extracellular traps (NETs)38 may occur and appears to have a phagocytic 
function. NETs are also regarded as a form of cell death different than apoptosis or necrosis. We are 
asking for approval to stain the neutrophil human cell to determine the level of NET formation under 
the conditions previously requested in the initial protocol. No extra specimens are requested. 
 
 
 
Innovation: 

 
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Revised March 13, 2014January 11, 2016 
The use of endotracheal intubation as a procedure to facilitate general anesthesia and provide airway 
protection in the intensive care unit and may result in a variety of airway injuries including sore 
throat. There is no current therapy universally accepted for prevention of sore throat despite a 
multitude of pharmacological and non-pharmacological interventions. Tracheal research is in itself 
innovative and analysis of local PMN activation in the context of acute tracheal injury represents a 
new approach in the quest to understand the pathophysiology of sore throat. Patient satisfaction after 
surgery is negatively influenced in part by sore throat. Identification of specific PMN surface 
receptors will provide therapeutic targets that can be used to modulate the inflammatory reaction and 
facilitate healing. The primary aim of this research is to improve understanding of the cellular 
mechanisms involved in sore throat following endotracheal intubation. If neutrophil activation 
patterns are correlated with occurrence of sore throat, more precise treatment(s) and preventive 
measurements can be taken to improve quality of patient care, satisfaction, safety and outcomes.  
This study may provide sufficient evidence to support new treatments such as anti-IL-6, anti-TNF-
alpha, Apyrase and other anti-inflammatory medications to coat the endotracheal tube and thus 
prevent sore throat and inflammation. 
 
Healthy Volunteers 
 
We found inflammation in the tracheal lavages of healthy humans indicating that neutrophil 
activation is occurring while the endotracheal tube is in place.  We hypothesize that the inflammation 
is mainly mediated by sterile products in particular necrotic tissue or cellular mitochondrial DNA 
resulting from local cell injury. Now we would like to recreate similar neutrophil response by looking 
in the blood of healthy volunteers and challenge the neutrophils with necrotic tissue or labeled 
mitochondrial DNA obtained from laboratory cultured cells and look for markers of neutrophil 
activation that also will include CD35/CD49b, connexin43 and the previously approved CD markers. 
By exploring the role of CD35/CD49b39 and possibly connexin4340 we could attempt to understand 
how the neutrophil cell respond to exposure to necrotic tissue and what possible mechanisms are 
involved in the responses. Determination of NET’s (neutrophil extracellular traps) will be also done 
as proposed previously for airway neutrophils. If recreation of the cellular injury in the blood 
neutrophil results in similar cellular changes as the ones seen in the trachea, then we will have a good 
human model to study airway inflammation. 
 
Approach 
 
2.1.1 Preliminary Data 
 
2.2 Objective 
The study is aimed at identifying neutrophil surface changes that occur during tracheal injury 
secondary to endotracheal intubation and to correlate them with the development of sore throat. 
 
2.3 Patient Selection 
 

 
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Revised March 13, 2014January 11, 2016 
2.3.1 Inclusion Criteria 
1. Ages 18 to 65 
2. American Society of Anesthesiologists Physical Status Classification (ASA) I and II as 
determined by the study physician as this is a subjective measure. 
3. Required to have endotracheal intubation 
4. Scheduled for short admission (<23 hrs), or ambulatory orthopedic surgery (ankle, 
shoulder, or extremity surgery); general surgery (exploratory laparotomy, 
cholecystectomy, hernia repairs); obstetric/gynecological surgery(total abdominal 
hysterectomy, bilateral salpingo-Oophorectomy); urologic surgery (nephrectomy, 
prostatectomy), lumbar spine surgery (discectomy, one or two level lumbar fusion) 
procedures. 
5.  
6. Provide informed consent. 
 
2.3.2 Exclusion Criteria 
1. Active pulmonary disease within 5 days prior to enrollment 
2. On immunosuppressive medications 
3. Previous tracheal surgery 
4. On azithromycin 
5. Diabetes 
6. Pregnancy 
7. Smoking history of less than 6 weeks prior to surgery 
8. Surgery or intubation within 5 days of surgery 
9. Planned surgical procedure involving neck or thoracic regions 
10. Active pulmonary disease 
11. Autoimmune disease (HIV, AIDS, Rheumatoid Arthritis 
12. Hepatitis B or C, or active Hepatitis A 
13.  Cancer 
 
Healthy Controls: 
Inclusion: 
1. 
Ages 18 to 65 
2. 
Provide informed consent 
 
2.4 Design and Procedures 
 
2.4.1 Study Design 
This study is observational and prospective.  
2.4.2 Minimization of Bias 
 

 
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DoCTR 
 
 
 
   
 
 
Revised March 13, 2014January 11, 2016 
There will be no specific gender or ethnic background for enrollment and the decision to place the 
endotracheal tube will be at the discretion of the attending anesthesiologist. Addition of 
CD35/CD49b and connexin43 will be done to determine neutrophil activity and subpopulation of 
neutrophils active during different conditions and may result in local inflammation or immune-
suppression. 
 
2.4.3 Pre-Study Period 
Subjects who are potential candidates for the study will be educated as to the study procedures, 
benefits, and potential risks.  Each subject who qualifies for entry into the study on the basis of 
inclusion/exclusion criteria, and completion of an informed consent will be assigned the next 
available patient number.  This indicates enrollment in the study. Subjects who drop out of the study 
prior to completing the study will be replaced by using the next available patient number. 
 
2.4.4 Study Period 
Subjects will participate from intubation time to the immediate post operative period.   
After the placement of the endotracheal tube (Hi-Lo™ Evac Mallinckrodt), tracheal lavages will be 
performed using 5 milliliters of sterile saline solution by a push/suction technique. Wall suction will 
be set to a minimum in order to minimize tracheal injury and to comply with standards of medical 
care. The same procedure for collection of tracheal sample will be repeated at the end of the surgical 
procedure,immediately prior to removal of the ETT. The specimen will be collected in a sterile 
container and placed in a laden ice container for transportation and immediate processing.  
 
A simultaneous blood sample of approx 5cc, will be collected by venipuncture or by sampling from 
intravenous or arterial line if available  at the end of the surgical procedure,immediately prior to 
removal of the ETT.  A total of approx 5cc blood will be collected for research purposes.  
 
Review of the medical record will be conducted for data acquisition of medical history, and 
preoperative blood test results to determine enrollment in the study according to the specified 
inclusion or exclusion criteria protocol. Physiologic and demographic measurements such as 
temperature, oxygenation, and blood pressure, gender, body mass index and coexisting diseases will 
be recorded as per anesthesia records. All data will be de- identified. All patients will be asked to 
respond to presence or absence of sore throat and its degree of intensity using a Visual Analogue 
Scale (VAS) sliding scale at 120 minutes post extubation and following a specific cognitive 
assessment performed by the recovery room nurses as part of their discharge protocol. Cognitive 
evaluation will include patient interview to determine the following: Orientation to time, place, 
person and situational (Why are you here?). 
 
Healthy volunteers 
A blood sample of approx 20cc, will be collected by venipuncture for research purposes to challenge 
the neutrophils with necrotic tissue obtained from laboratory cultured cells and look for markers of 
neutrophil activation that also will include CD35/CD49b and the previously approved CD markers 
 

 
Page 14 of 21 
 
 
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DoCTR 
 
 
 
   
 
 
Revised March 13, 2014January 11, 2016 
2.5 Observations and Measurements 
 
2.5.1 Lavage Processing 
Tracheal lavage fluid will be placed on ice and transported immediately to our laboratory. A sample 
of 0.25cc or less will be removed for cell count and differential count.  We will do total cell counts 
using tryptan blue to check viability of cells. Remaining whole fluid will be centrifuged at 1500 rpm 
for 15 minutes at 4° C and supernatants frozen in aliquots.  
 
2.5.2 Neutrophil assessment and purification 
Neutrophils will be counted in the tracheal lavage and peripheral blood with a HEMAVET analyzer 
(Drew Scientific). Neutrophils will be purified by immunomagnetic bead-mediated negative selection 
with biotin-labeled antibodies (e-biosciences) specific for CD11b, CD62L, CD54, CD32, CD88, and 
CD66b. Flow cytometry will be use for assessment of neutrophil surface receptor presence. 
 
2.5.3 Reactive Oxygen Species (R0S) determination 
Neutrophil production of ROS will be assessed with the fluoro probe dihydrorhodamine. 
 
2.5.4 Interleukin Assays 
Accumulation of interleukin 1 (IL-1), IL-4, IL-6, IL-8, IL-10, IL-17, IL-23p19, transformin growth 
factor 1 (TGF β-1) and tumor necrosis factor alpha (TNF-α) in culture will follow stimulation with 
formylated peptides by immunosorbent assay (ELISA) for human cytokines (R&D Systems, 
Minneapolis, MN). 
 
2.5.5 Toll-like Receptors (TLR) 
Determination of TLR-2, TLR-4, TLR-9 and mitochondrial DNA will be done by PCR.  
 
2.5.6 Neutrophil extracellular traps (NETs) formation 
Neutrophil cells will be stained and photographic documentation of the NET presence will be 
documented. 
 
2.5.7 Primary Outcome Measures 
The study is aimed at identifying neutrophil surface changes that occur during tracheal injury 
secondary to endotracheal intubation and to correlate them with the development of sore throat. 
 
2.5.8 Exploratory Secondary Outcome Measures 
To analyze the determine the presence and role of GMSTM1 in all the specimens collected and in the 
prospective human donors.  
 
Evaluate the role of autophagy 1 [ATG16L1] genetic polymorphisms of neutrophils in patients with 
and without postoperative airway inflammation or ventilator-associated pneumonia. ¶ 
 
2.5.9   Statistical Methods 

 
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DoCTR 
 
 
 
   
 
 
Revised March 13, 2014January 11, 2016 
The primary aim of this study is to describe the inflammatory changes that result in a neutrophil 
surface receptor expression unique to the interaction between the trachea and the ETT and result in 
sore throat. Blood samples will be obtained and neutrophil cells (PMN) will be isolated for analysis at 
the same time intervals as the tracheal lavage samples. Individual indicators of inflammation (PMN, 
cytokines, clusters of differentiation –CD, TLR’s as described above) will be collected at specific 
pre-determined times and will be compared to baseline values of these variables measured at time 
zero. We will compare neutrophil (PMN) changes between patients who developed sore throat and 
those who do not by using the Wilcoxon rank sum test, since we expect the data to be non-normally 
distributed.  
 
2.5.10   Sample Size 
With a sample size of 60 evaluable patients, we will have at least 80% power to detect large 
differences (Cohen’s D≥O.8) between the sore throat and non-sore throat groups with an alpha level 
of 0.05 and assuming that half of the participants report a sore throat following intubation. As our 
pilot study is exploratory, we did not inflate our sample size to account for multiple testing. We will 
confirm any identified associations found in our pilot study with a future larger study.  All subjects 
that undergo intubation attempts times 2 or more, will not be considered evaluable for the primary 
aim of this research.  However, these patients will be considered as a subset and of possible interest 
for analysis and outcome measures. 
 
Data analysis of the blood neutrophil changes occurring under different laboratory conditions will be 
analyzed using a Student-t test to compare them with changes observed in the injured airway 
neutrophils. A total of 25 evaluable patients will provide sufficient blood neutrophils to determine 
differences.   
 
3.0 Management of Intercurrent Events 
3.1 Adverse Experiences 
The investigator will closely monitor subjects for evidence of adverse events.  All adverse events will 
be reported and followed until satisfactory resolution.  The description of the adverse experience will 
include the time of onset, duration, intensity, etiology, relationship to the study procedures (none, 
unlikely, possible, probable, highly probable), and any follow up treatment if required. 
 
3.2 Premature Discontinuation 
If a subject withdraws from the study, the subject will be replaced in order to provide the required 
number of evaluable subjects.  Subjects will be withdrawn if the investigator decides that 
discontinuation is in the best interest of the subject, or the subject requests withdrawal from the study. 
 
3.3 Potential Risks 
 
3.3.1 Potential risks from ETT saline lavage 
By using sterile normal saline solution we minimized the impact of the irrigation on the trachea since 
the osmolality of the saline is similar to the osmolality of the serum. The collection of tracheal 

 
Page 16 of 21 
 
 
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agreement by the recipient that no unpublished information contained herein will be published or disclosed without prior written 
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DoCTR 
 
 
 
   
 
 
Revised March 13, 2014January 11, 2016 
secretions will be performed by trained personnel. Wall suction will be set to a minimum in order to 
minimize tracheal injury and to comply with standards of medical care. Although the endotracheal 
tube does not contain any particular medication, its presence may induce sore throat (reason for the 
study). The ET tube is being placed as standard of care for patients undergoing general anesthesia. 
The intrinsic risks related to the placement of the endotracheal tube are beyond the scope of our 
research. 
 
3.3.2 Potential risks from blood draw 
Discomfort, bruising, and/or bleeding at the site of the needle injection. Every attempt will be made 
to collect the blood sample from an indwelling venous or arterial catheter. All samples will be 
collected using standard medical techniques by trained personnel.  
 
3.3.3 Potential risk from loss of confidentiality 
One risk of participating in this study is that confidential information may be accidentally disclosed.  
We will use our best efforts to keep the information secure, and the risk of accidental disclosure is 
very small. All samples and health information will be de-identified and assigned a study ID number. 
Statisticians involved in the project will have access only to de-identified data for the purposes of 
analysis. The original informed consents will be kept by the PI and a copy will be given to all 
subjects. Neutrophil surface activation data samples will be kept confidentially. Any information 
obtained from medical records will be coded, with a key to the code linking code numbers to names 
kept at a separate location, under lock and key. 1) The link to identifiers will be destroyed at the end 
of the study. We have no evidence to suggest that testing will provide evidence of previously 
undiagnosed or unrecognized illness, or susceptibility to illness. 2) We will not use samples for any 
purpose other than to perform tracheal and blood neutrophil analysis. 3) Data will be stored under 
lock and key (office, file cabinet) and only the investigators will have access. If data are published, 
there will be no link to identifiers. Study data will not be revealed to any organization or individuals. 
 
3.4 Procedures to Minimize Potential Risks 
Study related procedures will be conducted in the operating room under the general supervision of the 
PI, a board-certified anesthesiologist, thoroughly trained and experienced in intubation, tracheal 
lavage, and blood collection. Subjects will be monitored through-out the study period by trained 
personnel. Subjects will be informed that participation is voluntary and they may refuse to participate 
and may withdraw from the study at any time without penalty.  Subjects will be told that in the event 
of a physical injury as the direct result of study procedures, they will be cared for by a member of the 
investigating team at no cost, within the limits of the Washington University compensation plan.  
 
3.5   Data and Safety Monitoring Plan 
The principal investigator will monitor the study for any adverse events and serious adverse events 
per the institutional guidelines. All unanticipated problems will be reported to the HRPO. Should 
there be a serious adverse event that occurs that increases the risks to the participants, the study will 
be stopped, an investigation will be conducted, and a findings report will be generated before the 
study is resumed. The specific monitoring plan for this investigation is commensurate with the risks 

 
Page 17 of 21 
 
 
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DoCTR 
 
 
 
   
 
 
Revised March 13, 2014January 11, 2016 
and the size and complexity of the investigations planned.  These risks, as described above, are small. 
Based on these considerations, we will utilize a monitoring plan that involves:  
(1) Engaging a colleague not involved in the study to serve in a monitoring capacity.  Based on the 
small size and low-risk, we envision only a third person (the colleague), not a full DSMB. 
(2) A specific plan for submitting Adverse Event Reports to the IRB.  That plan is detailed in the 
department’s SOP for Adverse Event Reporting (attached). 
 
4.  HUMAN SUBJECTS RESEARCH 
 
4.1 Protection of Human Subjects 
 
The study will be conducted under appropriate Washington University Institutional Review Board 
protocols and consent forms approvals.  The study will be conducted under the supervision of the PI, 
a Board-Certified and GCP-certified anesthesiologist, and a mentor with several years experience in 
the conduct of human volunteer studies. 
 
4.2 Sources of Materials 
Data will be collected from the subject’s electronic medical record.  Blood and tracheal specimens 
will be obtained for research purposes only. Patients will be asked to report presence or absence of sore 
throat and to score the severity of pain using a sliding VAS ruler.  
 
4.3 Recruitment and Informed Consent 
Subjects will be recruited from the PI’s patient population when they are admitted to the surgical 
holding area prior to ambulatory surgery. Healthy volunteers will be recruited using flyers posted 
around the medical school campus. Subjects will be provided verbal and written descriptions of the 
study aims, procedures, risks, and benefits, and will be required to give written informed consent.  A 
member of the investigative team provides all study descriptions, informed consent, and answers all 
questions.  No deception is required for the purposes of this study.  Subjects are informed verbally 
and in writing that participation is voluntary and they may refuse to participate and may withdraw 
from the study at any time without penalty. 
 
4.4 Potential Benefits of the Proposed Research to the Subjects and Others 
There is no immediate benefit to individual subjects in this study. This study seeks to identify 
neutrophil surface changes that may occur related to sore throat and may serve as reliable 
biomarker(s) for designing possible therapies that would benefit future patients. 
 
4.4.1 Importance of the knowledge to be gained  
The incidence of sore throat is significant and the pathophysiology is still unknown. There is no 
reliable way to diagnose or treat sore throat following placement of ETT. Many patients rate sore 
throat as a significant issue following general surgery and is viewed negatively by patients. If a 
patient requires endotracheal intubation for several days the inflammation documented in our 
previous studies may represent only the beginning of a more distal and severe form of respiratory 

 
Page 18 of 21 
 
 
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DoCTR 
 
 
 
   
 
 
Revised March 13, 2014January 11, 2016 
disease such as bronchitis, possible pneumonia. Finding and treating early sore throat may improve 
and prevent other respiratory disease. If our research demonstrates the surface changes in the 
neutrophils, we may be able to design an endotracheal tube coated with a specific anti-inflammatory 
receptor blocker and thus prevent occurrence of upper respiratory diseases. 
 
4.5 Inclusion of Women 
Studies and their advertisements actively encourage the participation of women in the research.  As a 
matter of operational policy, our studies of healthy volunteers routinely and deliberately include 
equivalent numbers of women and men. To ensure sufficient enrollment of women, we typically 
close enrollment to men once their quota has been filled.  This approach has been highly successful.   
 
4.6 Inclusion of Minorities 
All of our studies and their advertisements actively encourage the participation of minorities in 
research.  Our minority recruiting typically matches the demographic composition of the Washington 
University community from which subjects will be recruited (78% white, 21% Black, <1 % 
Hispanic).   However, based on the higher incidence of CYP2B6*6 allele in African-Americans, we 
anticipate a higher enrollment of minorities related to increased incidence of genotype.  
 
4.7 Inclusion of Children 
Children <18 yr will not be studied in this investigation.  
 
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agreement by the recipient that no unpublished information contained herein will be published or disclosed without prior written 
approval. 
 
DoCTR 
 
 
 
   
 
 
Revised March 13, 2014January 11, 2016 
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CONFIDENTIAL: This document is the intellectual property of Carlos Puyo, M.D.  Acceptance of this document constitutes the 
agreement by the recipient that no unpublished information contained herein will be published or disclosed without prior written 
approval. 
 
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Revised March 13, 2014January 11, 2016 
29. Gourlay T, Samartzis I, Stefanou D, Taylor K. Inflammatory response of rat and human 
neutrophils exposed to di-2-ethyl-hexyl)-phthalate-plasticized polyvinyl chloride. Artif Organs. 2003 
Mar;27(3):256-60. 
30. Vetrano AM, Laskin DL, archer F, et al. Inflammatory Effects of Phthalates in Neonatal 
Neutrophils. Pediatr Res. 2010 August; 68(2): 134-139. 
31. Caruso P, Denari S, et al. Saline instillation before tracheal suctioning decreases the incidence of 
ventilator-associated pneumonia. Crit Care Med 2009; 37:32-38. 
32. Fortunati E, Kazemir KM, Grutters JC, et al. Human Neutrophils switch to an activated 
phenotype after homing to the lung irrespective of inflammatory disease. Clinical and Experimental 
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35. Coppadoro A, et al, Modifying endotracheal tubes to prevent ventilator-associated pneumonia. 
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36. Pneumatics IA, Dragomans CK, Boutros DE. Ventilator–associated Pneumonia or Endotracheal 
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Q, Kuijpers TW, Wirawan E, et al. Cell Death and Differentiation (2011) 18, 581-588. 
39. Pliyev B K, Shmidt E I, Ivanova AV, Menshikov M. Circulating CD35_/CD49d+ neutrophils in 
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system. Biochimica et Biophysica Acta 1662 (2004) 102-112

 
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DoCTR 
 
 
 
   
 
 
Revised March 13, 2014January 11, 2016 
Appendix A 
Date 
 
Address 
 
 
To Whom It May Concern: 
 
On behalf of the research team at Washington University in Saint Louis School of Medicine and the 
Department of Anesthesiology and Critical Care, we would like to thank you for your participation in 
research study #  ___ - Sore throat secondary  to endotracheal intubation: the role of neutrophil 
activation  
 
If you have any questions about your participation in this research study, please feel free to contact 
Dr. Carlos Puyo at 314-747-0259 or e-mail: puyoc@wustl.edu. 
 
Thank you, 
 
 
 
Carlos Puyo, M.D., FCCP 
Assistant Professor  
Washington University Saint Louis 
Department of anesthesiology and Critical care