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Year : 2008  |  Volume : 52  |  Issue : 1  |  Page : 28 Table of Contents     

Tracheostomy In ICU: An Insight into the Present Concepts

Director (Academic) President, IACTA, Consultant & Professor Anaesthesia and Intensive Care, Narayana Hrudayalaya Institute of Medical Sciences, India

Date of Acceptance15-Dec-2007
Date of Web Publication19-Mar-2010

Correspondence Address:
Muralidhar K.
#258/A Bommasandra Industrial Area, Anekal Taluk, Bangalore-560099
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Source of Support: None, Conflict of Interest: None

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Tracheostomy is a commonly performed surgical procedure in the intensive care units. Indications for tracheostomy are mainly four-fold namely airway obstruction, aspiration of secretions, airway protection from aspiration and provision of mechani­cal ventilation. Anaesthesia technique used for tracheostomy is varied and is dictated by the general condition of the patient. Percutaneous tracheostomy is an alternative to the surgical approach that can be done at the bedside and has several advan­tages. Though a simple procedure, tracheostomy can be associated with a number of life-threatening complications like hypoxia, cardiac arrest, injury to structures immediately adjacent to the trachea, pneumothoax and haemothorax.

Keywords: Tracheostomy; Percutaneous tracheostomy; Permanent tracheostomy; Tube, tracheo-stomy; Emergency tracheostomy

How to cite this article:
K. M. Tracheostomy In ICU: An Insight into the Present Concepts. Indian J Anaesth 2008;52:28

How to cite this URL:
K. M. Tracheostomy In ICU: An Insight into the Present Concepts. Indian J Anaesth [serial online] 2008 [cited 2020 Oct 20];52:28. Available from: https://www.ijaweb.org/text.asp?2008/52/1/28/60595

   Introduction Top

Tracheostomy is one of the most frequently per-formed surgical procedures on critically ill patients re-quiring prolonged mechanical ventilation in the intensive care unit. It is performed in about 24% of all patients in intensive care units [1] . "Tracheotomy" is the procedure involving making incision over the trachea where as the term "Tracheostomy" is used to indicate a procedure involving incision on the trachea followed by insertion of a tube which maintains the patency of the opening in trachea either temporarily or permanently. Tracheostomy has several advantages over translaryngeal endotracheal intubation including lower airway resistance, smaller dead space, less movement of the tube within the trachea, greater patient comfort and more efficient suction. The relative merits of both the methods of airway access is highlighted in the [Table 1] below:

   Indications for tracheostomy Top

The following clinical scenarios illustrate the indi-cations for tracheostomy:

1.A 69-year female presented with a history of stiff-ness of joints, paraesthesia and weakness of both upper and lower limbs for 3 months. She was a known case of rheumatoid arthritis. CT scan of cervical spine revealed atlanto-axial subluxation and basilar invagination with significant spinal canal stenosis and spinal cord compres-sion. Preoperative medications included atenolol for hypertension and methotrexate for rheumatoid arthritis. She underwent foramen magnum decompression and fixa-tion of occipital C2 - 3 using a plate and rod system, screws and iliac crest grafting. Anaesthesia included propofol, rocuronium, fentanyl, isoflurane and mechani-cal ventilation (IPPV). At the end of surgery, the tra-chea was extubated after reversal of neuromuscular blockade. However, the patient developed respiratory failure and endotracheal intubation proved difficult due to cervical fixation and anterior larynx. Airway was maintained with laryngeal mask airway (LMA) and emergency tracheostomy was performed. [Figure 1] Indi-cation for tracheostomy: difficult airway, respiratory fail-ure and need for mechanical ventilation.

2.A 60-year female underwent off pump coronary artery bypass grafting (CABG). Postoperative recov-ery was delayed due to neurological deficits. CT scan revealed acute cerebral infarction involving both frontal lobes. Tracheostomy was done to protect airways from aspiration [Figure 2]. Indication for tracheostomy: airway protection from aspiration pneumonitis.

3. A 75-year male underwent CABG. Postopera-tive course was complicated by pneumonia. Tracheo-stomy was done for aspiration of secretions [Figure 3]. Indication for tracheostomy: broncho- pulmonary toilet and aspiration of secretions.

Thus the indications for tracheostomy include the following:

  1. Upper airway obstruction
  2. Prolonged mechanical ventilation
  3. Access for tracheal toilet
  4. Airway protection form aspiration of gastric / pha-ryngeal contents.

The presence of a difficult airway in patients re-quiring prolonged mechanical ventilation is an absolute indication for tracheostomy. Patients with difficult air-way include those with conditions such as maxillo-facial trauma, angioedema, obstructive upper airway tumors or other anatomical characteristics that would render translaryngeal intubation technically difficult to perform in the event of inadvertent airway loss. Patients needing prolonged ventilation undergo tracheostomy to facilitate ventilator weaning, diminish the incidence of serious in-fections, promote oral hygiene, pulmonary toilet, enhance patient comfort, provide airway security and in selected patients allow nutrition and speech.

   Anaesthesia for tracheostomy Top

Anaesthesia requirements are variable and are dictated by the state of patient′s sensorium and the nature and extent of co-morbid conditions. The procedure can be done safely with local infiltration supplemented with intravenous sedation / or narcotic drugs appropriate to the general condition of the patient. The advantage of this technique is that it allows for the administration of high levels of inspired oxygen. Inpatients with stable cardiovascular system general anaesthesia with or without supplemental local infiltration are satisfactory. Monitoring techniques are dictated by the extent of comorbidity but ECG, BP, pulse oximetry and end-tidal CO 2 monitoring is mandatory.

   Surgical procedure Top

The patient is placed in the supine position, the neck extended with the help of either a rolled towel or a small pillow placed beneath the shoulders [Figure 4]. The head is positioned in a ′head-ring′ for additional stability. These maneuvers provide for maximal surface exposure and, in most patients, bring the trachea from the intrathoracic to the cervical position. After appropriate preparation of the skin and surgical draping, the procedure is accom-plished through a short horizontal incision placed at the level of the second tracheal ring. The strap muscles are separated in the midline, and the thyroid isthmus is di-vided and sutured appropriately to obtain haemostasis. Counting down from the easily palpable cricoid cartilage makes specific identification of the location of the tra-cheal rings [Figure 5]. The second and third rings are opened vertically in the midline for access to the tra-chea. The tracheostomy tube should be placed so that it does not erode the ring and press against the cricoid cartilage. In addition, the opening should not be placed too low, or the tip of the tube and its cuff will be too close to the carina. Low placement of the tracheostomy tube is also hazardous because the innominate artery crosses anteriorly to the trachea low in the neck and the possibility of erosion into the vessel by either the cuff or tip of the tracheostomy tube exists. Segments of the tra-chea should not be removed because this might lead to a greater loss of tracheal wall stability and predispose the remaining segments to stenosis, once healing is accom-plished after removal of the tube. The lateral tracheal walls are retracted and the appropriately sized tracheo-stomy tube is inserted into the airway after slow withdrawal of the previously placed oral or nasal endotra-cheal tube to a more proximal position in the trachea. Once the tracheostomy tube is positioned and an ad-equate airway is demonstrated through positive pressure ventilation and visual inspection of chest wall expansion, confirmed by the end-tidal CO 2 tracing the previously placed endotracheal tube is removed from the trachea. The wound is closed appropriately, skin sutures are placed through the flange ends, and a tie is secured around the neck.

   Timing of tracheostomy in acute respiratory failure Top

The advantages of performing early tracheostomy in patients needing long-term ventilation are mentioned in the [Table 2]:

There are several reasons why a tracheostomy may facilitate weaning from mechanical ventilation. Resis-tance to airflow in an artificial airway is proportional to air turbulence, tube diameter, and tube length. Air turbu-lence is increased in the presence of extrinsic compres-sion and inspissated secretions. Because of its rigid de-sign, shorter length, and removable inner cannula (to al-low for evacuation of secretions), airflow resistance and associated work of breathing should theoretically be less with tracheostomies relative to endotracheal tubes. Fur-thermore, the presence of a tracheostomy may allow clinicians to be more aggressive about weaning patients from mechanical ventilation. Specifically, if a patient with a tracheostomy tube in place does not tolerate liberation from mechanical ventilation, he or she may be recon-nected to the ventilator without difficulty. In contrast, if a patient who is translaryngeally intubated does not tol-erate extubation, he or she must be sedated and re-intu-bated. This might represent a potential barrier to extu-bation in patients who are of a marginal pulmonary sta-tus. Finally, efforts to determine the relative advantages of tracheostomy and translaryngeal intubation with re-spect to aspiration are inconclusive. However, if the pres-ence of a tracheostomy does translate into earlier lib-eration from mechanical ventilation, one might expect the incidence of ventilator-associated pneumonia in this group to be lower. A clinical study that adequately ad-dresses the question as to optimal timing of tracheostomy in the setting of prolonged mechanical ventilation must have a homogeneous patient population, protocols in place for ventilator weaning and other facets of clinical man-agement, and well-defined endpoints. Given the lack of evidence on which to base decision-making on this is-sue, the following guidelines have been formulated [2].

  • For patients in whom the need for ventilatory sup-port is anticipated to be less than 10 days, translaryngeal intubation is preferred.
  • If the need for ventilatory support is anticipated to exceed 21 days, a tracheostomy is preferred.
  • When the anticipated need for mechanical ventila-tion is unclear, daily assessment is required to deter-mine when conversion to tracheostomy is indicated.

   Percutaneous tracheostomy Top

Percutaneous tracheostomy (PCT), which has been used to some extent for the past 50 years, is an alternative to the open surgical approach. The advan-tages and different techniques of percutaneous tracheo-stomy are highlighted in the [Table 3]:

The most popular technique, known as percutane-ous dilatational tracheostomy (PDT), was originally de-scribed by Ciaglia in 1985 [3] . In principle, the technique employs the use of serial dilators introduced into the tra-chea over a guide wire and is generally done with bron-choscope visualization. Modifications to improve this tech-nique have included design of special dilating forceps and use of a single-tapered dilator rather than multiple-sized ones. Several commercial PDT kits are currently available, providing all of the necessary equipment for efficient and expedient application. The steps in perform-ing percutaneous tracheostomy using "Rhino-horn" is depicted in [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13].

It is likely that the general trend toward minimally invasive surgery and the increase in procedure done by nonsurgical, interventional specialists will continue to promote use of this beside procedure Advocates of this technique frequently focus on use in critically ill patients in an intensive care unit (ICU) setting. Obvious advan-tages in this setting include avoidance of inherent risks associated with transport to and from the operating room, availability of existing monitoring, and cost savings with respect to operating room charges and anaesthesia fees. Although multiple published studies have attempted to compare the risk and benefits of the percutaneous procedure with the open procedure, problems with study design and the multiple variables that exist in patient populations, techniques, and operator experience create prob-lems in analysis of outcome [4],[5] . Two recently published meta-analyses summarize the potential difference in complications with both approaches. Stomal infection, bleeding, and trauma to structures adjacent to the tra-chea are some of the most commonly cited complica-tions. The following guidelines are taken into account when the question of whether surgical or percutaneous approach is to be decided.

Surgical Tracheostomy preferable to PCT in the following situations:

  • Coagulation abnormality
  • High level of ventilatory support (FiO2> .7 & PEEP > 10 cm H2O)
  • Unstable / Fragile cervical spine
  • Neck injury
  • Unfavorable neck anatomy (previous surgery, tu-mor etc.)
  • Obesity

   Tracheostomy tubes Top

Most tracheostomy tubes are manufactured from polyvinyl chloride, silicone, a combination of these mate-rials, or metal [Figure 14] and [Figure 15]. They are available in either single-lumen (no removable inner cannula) or dual-lu-men (removable inner cannula) configurations. The pur-pose of the removable inner cannula is to facilitate clean-ing of inspissated secretions that may lead to tube occlu-sion. Because silicone is relatively secretion resistant, tubes manufactured from this material frequently do not have an inner cannula. Tracheostomy tubes are avail-able with and without cuffs (balloon surrounding the outer cannula). The purpose of the cuff is to maintain a seal between the tube and the trachea sufficient to prevent escape of air from around the tracheostomy tube during mechanical ventilation (e.g., cuff leak). Furthermore, the cuff minimizes but does not prevent aspiration. Tracheo-stomy tubes with foam cuffs conform to a patient′s tra-chea and remain consistently inflated at low pressure. These tubes are indicated in patients who have sustained damage from excessive cuff pressure (e.g, tracheomalacia). Once a cuffed tracheostomy tube is no longer required, that is, the patient no longer requires mechanical ventilatory support and is not considered on aspiration risk, the cuffed tube is exchanged for a cuffless tube. Tracheostomy caps are generally provided with tracheostomy tubes for use in the decannulation process. Fenestrated tubes are used to promote speech and are generally used in individuals who tolerate liberation from mechanical ventilation for varying periods. Fenestrated tubes have an opening or openings on their superior as-pect such that when the inner cannula is removed, the cuff deflated, and the external orifice occluded (e.g., with a Passey-Muir type valve), air can pass the vocal cords allowing phonation.

   Minitracheostomy Top

Sputum retention, by definition, exists whenever a patient is incapable of clearing his own tracheobronchial secretions. Diagnosis of this condition is essentially clini-cal and is based on clinical evidence of respiratory dis-tress with rapid, shallow and bubbly respiration [6] . Chest physiotherapy when used early and frequently aided with cough maneuvers prevents sputum retention. If this fails, secretions have to be removed actively by suction. Minitracheostomy provides a permanent access to the trachea for suction while avoiding the disadvantages of the conventional methods. The procedure of minitracheostomy is simple and consists of percutane-ous tracheal cannulation using a 4 mm portex paediatric endotracheal tube inserted through a 1 cm incision in the cricothyroid membrane. The patient is positioned as for a routine tracheostomy with extension of the head and the neck. After cleansing the skin and local infiltration with 1% lidocaine, a midline vertical 1 cm stab incision is made through the cricothyroid membrane. The introducer is then passed through the stab incision into the trachea. The cannula is passed over the introducer and into the trachea; the introducer is then removed. The wings on the cannula are bent laterally and then sutured to the skin. The relative advantages of minitracheostomy over the conventional methods are: (1) It allows constant tra-cheal access for suction, (2) All natural mechanisms are retained; the function of the glottis is preserved. Patients retain an expulsive cough even if only by reflex response to suction catheter; minitracheostomy thus supplements the intrinsic clearing mechanisms, (3) Used early, it can prevent the onset of respiratory failure, (4) It is a simple technique to perform, (5) It has quick healing with mini-mal scarring, (6) It can be kept isolated from a median sternotomy wound, (7) Retention of voice and the ability to communicate confers significant advantages (8) Can be used for administration of humidified air / O 2 mixture or nebulised drugs (9) Can be used to obtain samples of sputum without salivary contamination for bacteriologi-cal studies, (10) It can be used in the management of acute airway obstruction and in certain forms of faciomaxillary surgery and (11) It can be used as a route for high frequency jet ventilation and intra-airway pres-sure monitoring [7] .

   Decannulation Top

Patients who remain stable for 24 to 48 hours after discontinuation of mechanical ventilation may be evalu-ated for decannulation. The patient′s ability to protect the airway should be assessed for 24 hours by deflating the tracheostomy tube balloon and observing for signs of aspiration. If aspiration is present, laryngoscopic ex-amination should be performed. Deflating the tracheostomy tube balloon and occluding the tracheostomy tube can assess airway for strictures and adequacy of the native airway. Patients who are able to breathe around a capped and deflated. No.8 tracheostomy tube most likely have adequate respiratory reserve and a sufficiently preserved native airway to tolerate decannulation. Pa-tients who have difficulty in breathing around a capped No.8 tube should be reassessed with a capped No.7 tra-cheostomy tube. Successful breathing with a capped and deflated No.7 tube in place suggests that a patient will tolerate decannulation. Patients who fail breathing trials with capped tracheostomy tubes should undergo laryngo-scopic evaluation to exclude the presence of tracheal steno-sis. Many patients recovering from long-term mechanical ventilatory support may have normal airways but fail breathing around a capped No.7 or No.8 tracheostomy tube because of limited ventilatory reserve (e.g., neuro-muscular disease or underlying chronic obstructive pul-monary disease). These patients may benefit from "downsizing" of the tracheostomy stoma using progres-sively smaller cuff-less tracheostomy tubes with intermit-tent capping using stomal obturators. Tracheostomy tubes with foam cuffs should not be used for decannulation tri-als because these cuffs spontaneously reinflate, making the assessment of airway stenosis difficult.

   Complications Top

A variety of complications resulting from tracheo-stomy placement have been described. A brief discus-sion of common complications occurring in the critical care setting and their management follows.

Complications of tracheostomy

Intra operative

  • Bleeding
  • Damage to adjacent structures
  • False passage

Post procedural

  • Cuff leak
  • Tube occlusion
  • Tube dislodgement
  • Tracheo-esophageal fistula
  • Tracheo-innominate fistula
  • Tracheal stenosis

   Intraoperative complications Top

Intraoperative complications generally occur as a result of anaesthesia, underlying disease. Surgical com-plications generally fall into three major categories, in-cluding hemorrhage, injury to structures adjacent to the trachea, and no airway cannula. Bleeding from the sur-gical incision is usually controlled easily but can be com-plicated by the difficulty of exposure. Vascular struc-tures (such as the thyroid isthmus) might bleed easily when divided for exposure. Injuries to adjacent struc-tures include damage to recurrent laryngeal nerves, en-trance into major vessels, and rare (but possible) lacera-tion of the esophagus. The inability to cannulate the tra-chea is possible because of inadequate surgical expo-sure, an inability to bring the trachea to a superficial lo-cation, or selection of a tracheostomy tube too large to fit into the tracheal stoma.

   Cuffleaks Top

Cuff leak is a commonly encountered problem in patients with tracheostomies and may be manifested by either an audible leak around the tracheostomy tube or loss of returned volume in mechanically ventilated breaths. A mechanical problem with the tracheostomy tube should first be excluded by determining that when the cuff is inflated it does not leak air. A malfunctioning tracheostomy tube requires exchange. Once tracheo-stomy tube malfunction is excluded, the most common cause of cuff leak is tracheomalacia with resulting dila-tion adjacent to the tracheostomy tube cuff. This is par-ticularly common in patients who have been maintained on mechanical ventilation for extended periods. It should not be treated by hyperinflating the tracheostomy tube cuff in an effort to achieve total occlusion, in that this will result in further dilation of the trachea and may lead to mucosal ischemia. If the cuff leak is well tolerated, such that the ability to ventilate the patient is not com-promised, we recommend maintaining the tracheostomy tube in place at the appropriate inflation pressure (e.g., 20 to 25 mm Hg). Conversely, if the cuff leak is suffi-cient so as to impair gas exchange, consideration should be given to exchanging the tracheostomy tube for either a larger size or for a tracheostomy tube design that in-corporates a large-volume, low-pressure cuff (e.g., a foam cuffed tracheostomy tube).

   Tube occlusion Top

A frequently encountered problem in patients with tracheostomies is tracheostomy occlusion. This is typi-cally manifested by either high airway pressures or inabil-ity to pass a suctioning catheter. Tracheostomy tube oc-clusion is frequently the result of inspissated secretions. Many commonly used tube designs have a removable in-ner cannula to facilitate cleaning of the inner portion of the tracheostomy tube. A second common cause of tra-cheostomy tube occlusion is tube malpositioning, such that the end of the tracheostomy tube abuts the tracheal wall or the tube has migrated such that its tip resides in the pretracheal tissues. If tracheostomy malpositioning is sus-pected, the operating surgeon should assist in assessing it for either reinsertion or use of another tube design.

   Tube dislodgement Top

Although dislodgment of the tracheostomy tube may occur at any time after tracheostomy placement, this complication is most problematic in the immediate post-operative period, before the tracheostomy tract has ma-tured. Factors predisposing to tracheostomy tube dis-lodgment include an inadequately secured tube, exces-sive coughing, and patient agitation. Tracheostomy tube dislodgement should be suspected when a patient is able to speak immediately after tracheostomy placement, the airway becomes obstructed, or respiratory distress de-velops. Because it is generally technically difficult to reinsert the tracheostomy tube in this situation, the au-thors recommend that the airway be reestablished by means of translaryngeal intubation. The tracheostomy should then be reinserted in the operating room with ap-propriate anaesthetic assistance, lighting, and instrumen-tation. If tracheostomy tube dislodgement occurs once the tracheostomy track is sufficiently mature (i.e., the tracheostomy track is at least 1 week old), it is generally technically feasible to reinsert the tracheostomy tube at the patient′s bedsides

   Tracheoesophageal fistula Top

The development of tracheoesophageal fistulas af-ter tracheostomy is rare, occurring in less than 1% of pa-tients and is typically the result of pressure necrosis of the tracheal and esophageal mucosa from the tracheostomy cuff. A number of potential risk factors have been re-ported (e.g., high airway pressures, excessive cuff infla-tion pressures, the use of nasogastric tubes, excessive tra-cheostomy tube movement). Clinical manifestations are nonspecific and include excessive tracheal secretions, coughing, and gastric distention. The presence of a tra-cheoesophageal fistula can be demonstrated on fiberoptic examination after removal or retraction of the tracheo-stomy tube. Because the use of fiberoptic examination alone is insensitive, it should be combined with an enter-ally contrasted esophageal evaluation if clinical suspicion exists (e.g., water-soluble contrast swallow or computed tomography). Tracheoesophageal fistula requires surgi-cal repair. Temporizing measures include positioning of an endotracheal tube cuff below the level of the fistula to limit aspiration, removal of nasogastric tubes, and place-ment of feeding gastrostomy tubes.

   Tracheoinnominate artery fistula Top

Tracheoinnominate artery fistula likewise is a rare complication after tracheostomy formation and theoreti-cally results from pressure necrosis or injury to the tra-chea adjacent to the course of the innominate artery. A number of risk factors have been postulated, including excessive tube movement, aberrant innominate artery anatomy, use of an excessively long or curved tracheo-stomy tube that erodes through the tracheal wall, infe-rior positioning of the tracheostomy tube, tracheal infec-tion, and corticosteroid therapy. Tracheoinnominate ar-tery fistula may become apparent as quickly as a few days or as late as several months after tracheostomy placement. The classic presentation is of a "sentinel hem-orrhage" in which a large volume of blood emanates from the tracheostomy tube. Fiber optic examination to evaluate for the presence of tracheoinnominate artery fistula should be performed in the operating room in the event that airway manipulation results in massive hem-orrhage. Temporizing measures in patients who develop massive bleeding include hyperinflation of the tracheo-stomy cuff, insertion of an endotracheal tube through the tracheostomy stoma in an effort to tamponade bleed-ing, or translaryngeal intubation and digital compression of the bleeding site through the tracheostomy stoma. Definitive repair entails median sternotomy, ligation of the innominate artery and generous drainage of the me-diastinum.

   Acknowledgement Top

The author acknowledges with thanks that parts of the text are derived from Wilson RS, Tracheostomy and tracheal resection and reconstruction, Chapter-10, in Thoracic Anaesthesia, Kaplan(ed), 6 th edition, 2003 & Freeman BD and Buchman TG, Indications and man-agement of tracheostomy, Chapter-72 Abraham F and Kochnek V(ed) in Text book of Critical care, 5 th edition, 2005. [Figure 16]

   References Top

1.1. Esteban A, Anzueto A, Alia I, et al. How is mechanical ventila­tion employed in the intensive care unit? An international uti­lization review, Am J Respir Crit Care Med 2000, 161:1450­-1458.  Back to cited text no. 1      
2.2.Consensus conference on artificial airways in patients receiv­ing mechanical ventilation. Chest 1989; 96:178-180.  Back to cited text no. 2      
3.3. Ciaglia P, Fishing R, Synice C. Elective percutaneous dilata­tional tracheostomy dilatational trachestomy; a new simple bedside procedure, Chest 1985;87:715.  Back to cited text no. 3      
4.4. Dulgueriv P, Gysin C, Perneger TV, et al. Percutaneous or sur­gical tracheostomy; a meta-analysis, Crti Care Med 1999; 27:1617.  Back to cited text no. 4      
5.5. Freeman BD, Isabella K, Lin N, et al. A meta - analysis of prospective trials comparing percutaneous and surgical tra­cheostomy in critically ill patients, Chest 2000;118:1412.  Back to cited text no. 5      
6.6. Matthews HR, Fischer BJ, Smith BE,et al. Minitracheostomy, a new delivery system for jet ventilation. J Thorac Cardiovasc. Surg 1986;92: 673.  Back to cited text no. 6      
7.7. Rao M, Muralidhar K. Treatment of sputum retention with minitracheotomy in postoperative cardiac surgical patients. J Anaesth Clin Pharmacol 1988;4:131-135.  Back to cited text no. 7      


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16]

  [Table 1], [Table 2], [Table 3]


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