All patients admitted to the multidisciplinary intensive care unit (ICU) of the EOC Regional Hospital "La Carità" in Locarno, Switzerland, between 2002 and 2004 were screened for eligibility. Patients with ALI or ARDS of different causes and with ACLE requiring immediate intubation and mechanical ventilatory support were enrolled (n = 54; Figure 1). Patients with ALI/ARDS were identified by the American-European Consensus Conference definitions [1]. The clinical diagnosis of ACLE was confirmed by reviewing patient records and chest radiographs, recent medical history, and echocardiography or pulmonary artery catheter if the diagnosis was not clear. ACLE was classified as acute exacerbation of congestive heart failure, acute coronary syndrome or exacerbation of diastolic left ventricular dysfunction. Patients were excluded if they had known HIV infection, immunodeficiency necessitating granulocyte colony stimulating factor, ALI/ARDS after thoracic surgery, and mixed causes of pulmonary oedema with elements of both ALI/ARDS and elevated hydrostatic pressure (n = 24). Finally, 30 mechanically ventilated patients met the eligibility criteria (Figure 1). Patients were intubated with oral endotracheal tubes with an internal diameter of 8 mm or more.
This study was approved by the Committee of Human Research of the Canton of Ticino, Switzerland, and informed written consent was obtained from each patient's next of kin.
Clinical data
Clinical, physiological and biological data at the time of fluid sampling and throughout the hospital course were recorded using a standardised data collection form. The Simplified Acute Physiology Score II (SAPS II) [11] and the Lung Injury Score (LIS) [12] were calculated. Outcome variables included ICU and hospital mortality and length of stay (LOS) in the ICU.
Stable patients were sedated at the time of fluid sampling with midazolam and/or propofol. Ventilatory support in patients with ALI/ARDS was carried out in accordance with the ARDS Network criteria for a protective lung strategy [13]. Patients with ACLE were ventilated with a plateau pressure (Pplat) limit of 30 cmH2O and a pressure-controlled or volume-controlled mode. During sampling with the mini-BAL catheter, arterial oxygen saturation (SpO2), haemodynamics (heart rate (HR), systemic arterial pressure (SAP)) and ventilatory variables (expiratory tidal volume (Vt), minute volume (VE), auto PEEP, peak pressure (Ppeak), and Pplat) were recorded. At the time of fluid collection, patients were treated with vasoactive agents, diuretics, antiarrhythmic agents, antibiotics and fluids (mainly sodium chloride 0.9%). None of the patients were treated with inhaled beta-adrenergic agonists before the sampling procedures.
Collection of samples
In order to enhance the comparison of the two methods, lung samples with s-Cath and mini-BAL were obtained early in the course of ALI/ARDS and ACLE (within one hour of intubation for the s-Cath and four hours for the mini-BAL).
s-Cath
Samples of distal pulmonary oedema fluid were collected without saline instillation by two of the authors (GC, GD) or by trained ICU nurses, following the method previously described by Matthay and co-workers [4, 5].
A 14-French (Fr) gauge tracheal s-Cath was blindly advanced through the silicone rubber diaphragm of the swivel adapter of the endotracheal tube into a wedge position in a distal bronchus. Undiluted fluid was then aspirated into a suction trap by gentle suction and stored for less than four hours at 4°C before processing. If the sample was sticky from airway mucus, a small amount (0.2 ml) of sodium citrate was added. The resulting new dilution factor was taken into consideration for the protein content measurements. The collection procedure lasted less than two minutes and was performed without complications in all patients. No modification of ventilatory settings was necessary during the s-Cath procedure.
mini-BAL
Mini-BAL was performed by means of a 16-Fr 5 mm outer diameter catheter introduced through a swivel adapter to allow maintenance of PEEP and to set VE (BAL Cath, Ballard Medical Products, Draper, UT, USA). By means of the external oxygen port, which allows the catheter to be directed, the 12-Fr inner catheter was advanced until a slight resistance was felt, indicating a wedged position. In three patients, the correct peripheral position of the tip was confirmed by fluoroscopy.
Lavage was performed with 30 ml aliquots of sterile saline, with the goal of instilling a total of 150 ml in five separate aliquots. After each aliquot, a gentle manual suction was applied to recover the instilled fluid. Fluid was kept in specimen traps and immediately processed in the laboratory. Dwell time was as short as possible and the whole procedure lasted less than 15 minutes after the instillation of the first aliquot. The patient's stability was monitored during this procedure by recording SpO2, HR, SAP, Vt, VE, auto-PEEP, Ppeak and Pplat. Arterial blood gas analysis was performed before and 30 minutes after the mini-BAL procedure.
Patients were pre-oxygenated with 100% fraction of inspired oxygen (FiO2) 15 minutes prior to sampling. This oxygen concentration was maintained during the sampling collection and for up to 30 minutes after removing the catheter. Then, if SpO2 was stable, the pre-BAL FiO2 was progressively restored over 30 to 60 minutes. The small 5 mm outer mini-BAL catheter diameter made it possible to maintain the pre-procedure ventilatory settings in most patients during the entire sampling collection [7]; the maintenance of the settings enabled analysis of ventilatory variables (pressures, blood gas) during and after the procedure. A peripheral blood specimen was collected from each patient at the time of the mini-BAL procedure. The mini-BAL procedure was not performed in eight patients because of haemoptysis, major cardiovascular instability or extreme hypoxaemia (partial pressure of oxygen in arterial blood (PaO2)/FiO2 < 100 with 100% oxygen). During the mini-BAL procedure, 5 of 22 patients experienced minor bronchial bleeding and the procedure was stopped prematurely.
Measurements
Oedema fluid obtained by means of the s-Cath was filtered through a 100 μm nylon cell strainer (Falcon 2360, Becton Dickinson, Frankling Lakes, NJ, USA). One aliquot (200 μl) was used for cell count (white blood cells (WBCs) and red blood cells (RBCs) respectively, including cell differential), with a Sysmex NE 1500 and the Sysmex K 1000 hematocytometer (Sysmex Europe GmbH, Norderstedt, Germany). Total protein concentration was measured after centrifugation by the Biuret technique. After recording the total volume of mini-BAL fluid, we filtered it through a 100 μm nylon cell strainer; at least 15 ml of the filtered solution was used for measurement of total and differential leukocyte counts. Cell count (WBC, RBC) was performed with a Sysmex NE 1500 and a Sysmex K 1000 hematocytometer. A centrifuged portion of mini-BAL fluid was used for measurement of total protein (Biuret method). The protein content was computed, after centrifugation, by taking into account the total BAL fluid volume for a given patient. The same strategy was used for all patients. The plasma total protein concentration was measured in duplicate by the Biuret method. A protein concentration ratio of oedema fluid:plasma was calculated.
Statistical analysis
Data are reported as means ± standard deviation or as medians and ranges. Comparison between groups was performed using the non-parametric Mann-Whitney-U test; normally distributed variables were compared by using the unpaired Student t-test. Continuous variables (variations of respiratory and haemodynamic variables during mini-BAL) were compared by using Student t-test, Wilcoxon signed rank test, analysis of variance or Student-Newman-Keuls test. Categorical variables were compared by using chi-squared analysis or Fisher's exact test. Finally, Bland-Altman plots [14] were used for assessing the mean bias and the limits of agreement between the two sampling techniques, using protein content and neutrophil percentage.