Supplementary Appendix

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This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Gattinoni L, Caironi P, Cressoni M, et al. Lung recruitment in patients with the acute respiratory distress syndrome. N Engl J Med 2006;354:1775-86.

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Lung recruitment in patients

with Acute Respiratory Distress Syndrome

Luciano Gattinoni*, M.D., F.R.C.P., Pietro Caironi*, M.D., Massimo Cressoni*, M.D., Davide Chiumello*, M.D., V. Marco Ranieri†, M.D., Michael Quintel‡, M.D., Ph.D., Sebastiano Russo‡, M.D., Nicolò Patroniti§, M.D., Rodrigo Cornejo¶, M.D., Guillermo Bugedo¶, M.D.

* Istituto di Anestesia e Rianimazione, Fondazione IRCCS – “Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena” di Milano; Università degli Studi di Milano, Italy;

† Dipartimento di Anestesia, Azienda Ospedaliera S. Giovanni Battista-Molinette, Università degli Studi di Torino, Italy;

‡ Anaesthesiologie II, Operative Intensivmedizin, Universitatsklinikum Gottingen, Gottingen, Germany;

§ Dipartimento di Medicina Perioperatoria e Terapia Intensiva, Azienda Ospedaliera S. Gerardo di Monza; Università degli Studi Milano-Bicocca, Italy;

¶ Departamentos de Anestesiologia y Medicina Intensiva, Facultad de Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile.

1) Additional methods (page 2 – 9) 2) Additional results (page 10 – 48)

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A

DDITIONAL METHODS

Approval of the study protocol and patient consent

The study was approved by the Institutional Review Boards of each participating Institutions (Istituto di Anestesia e Rianimazione, Fondazione IRCCS – “Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena” di Milano, Università degli Studi di Milano, Italy; Dipartimento di Anestesia, Azienda Ospedaliera S. Giovanni Battista-Molinette, Università degli Studi di Torino, Italy; Anesthesiologie II, Operative Intensivmedizin, Universitatsklinikum Gottingen, Gottingen, Germany; Departmentos de Anestesiologia y Medicina Intensiva, Facultad de Medicina,

Pontificia Universidad Catolica de Chile, Santiago, Chile). Patient consent was obtained according to the national regulations of each participating Institutions. As the patients were incompetent, patient consent in Italy was delayed according to the Italian regulations (“delayed consent” [1]). The family was informed of the study (although not required by the law) and the study was performed. As soon as competent, each patient was fully informed on what had been done, and a written permission of using data collected was obtained.

Enrollment

To take into account the availability of the CT-scan facility and the sufficient time to stabilize the patients in order to perform the study in a safe condition, the experimental protocol did not include any time-limitation to perform the study after the diagnosis of ALI/ARDS. As an

example, if the hemodynamic conditions of the patient were not stable, the study was postponed. Indeed, the time between the onset of ALI/ARDS and the study varied from 1 to 21 days (median 4 days, mean 5±5 days). Of note, the length of the time-period between the intubation and the day of the study did not appear to have any influence on the results observed in our study population (see pages 44 to 48 of the Supplementary Appendix).

Comparison groups

Data for comparison groups were obtained at five different Hospitals (Istituto di Anestesia e Rianimazione, Fondazione IRCCS – “Ospedale Maggiore Policlinico, Mangiagalli, Regina

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Elena” di Milano, Università degli Studi di Milano, Italy; I° Servizio di Anestesia e

Rianimazione, Ospedale Luigi Sacco di Milano, Italy; Dipartimento di Medicina Perioperatoria e Terapia Intensiva, Azienda Ospedaliera S. Gerardo di Monza, Università degli Studi Milano-Bicocca, Italy; Dipartimento di Anestesia e Rianimazione, Ospedale Riuniti di Bergamo,

Università degli Studi Milano-Bicocca, Italy; Dipartimento Emergenza Urgenza, Ospedale Civile di Legnano, Italy). From hospital databases, a total of 63 patients with lung injury other than ALI/ARDS, who underwent from 2001 and 2005 a whole lung CT-scan, and in which CT-scan data were available, were first selected. Among them, 62 patients were affected by pneumonia, and 1 patients by congestive heart failure. As not directly affected by a primary lung disease, the patient with congestive heart failure was excluded from the subsequent analysis. Among the remaining population, 28 patients were affected by bilateral pneumonia, and 34 patients by unilateral pneumonia, as diagnosed by the attending physician of the Emergency Unit. Within the group of patients with bilateral pneumonia, 15 patients had a PaO2/FIO2 value greater than

300 mmHg, not presenting inclusion criteria for ALI/ARDS; in contrast, the remaining 13

patients did actually meet the criteria for ALI/ARDS diagnosis (PaO2/FIO2 less than 300 mmHg),

but were not classified as such by the attending physician of the Emergency Unit. Therefore, to avoid any possible confusion and potential overlapping between patients with ALI/ARDS, especially the less severe ones, and patients with bilateral pneumonia, only patients with unilateral pneumonia (n=34) were selected and included in the comparison group. To ascertain the diagnosis of “unilateral pneumonia”, the ratio between the amount of non-aerated lung tissue of the affected lung and the total amount of non-aerated lung tissue was calculated for each patients, i.e. both patients with bilateral and patients with unilateral pneumonia, and only patients with a ratio greater than 0.7 were at first included. The diagnosis of unilateral pneumonia was then re-confirmed in each patients by visual examination of the whole lung CT-scan.

Data of patients with healthy lungs were obtained from the hospital database of the Istituto di Anestesia e Rianimazione, Fondazione IRCCS – “Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena” di Milano, Università degli Studi di Milano, Italy. Thirty-nine patients who underwent a whole lung CT-scan for general clinical assessment were retrieved and included in the comparison group.

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Calculations of physiological variables

The following equations were used for the computation of physiological respiratory variables.

1) Dead-space fraction (percent of tidal-volume):

2 2 2 PaCO CO P PaCO space dead = − E

where PaCO2 is the arterial partial pressure of carbon dioxide, and PECO2 is the mixed expired

partial pressure of carbon dioxide. This variable was automatically computed by the CO2SMO

monitor (Novametrix, Wallingford, CT), and was obtained in 48 patients, as only in these patients PECO2 was measured.

2) Alveolar-dead space fraction (percent of tidal-volume):

2 2 2 PaCO CO P PaCO space dead alveolar = − ET

where PaCO2 is the arterial partial pressure of carbon dioxide, and PETCO2 is the end-tidal partial

pressure of carbon dioxide. This measurement was obtained in 55 patients, as only in these patients PETCO2 was measured, and was automatically computed by the CO2SMO monitor

(Novametrix, Wallingford, CT).

3) Right-to-left intrapulmonary shunt fraction (percent of cardiac output):

2 2 2 2 CvO CcO CaO CcO fraction shunt − − =

where CcO2 is the capillary oxygen content, CaO2 is the arterial oxygen content, and CvO2 is the

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The capillary oxygen content (CcO2) was computed as:

[

2*0.003

]

*100%*1.39

2 P O Hb

CcO = A +

where PAO₂ is the alveolar partial pressure of oxygen, and Hb is the blood concentration of hemoglobin.

The arterial oxygen content (CaO2) was computed as:

[

₂ *0.003

]

* ₂*1.39

2 PaO Hb SaO

CaO = +

where PaO2 is the arterial partial pressure of oxygen, Hb is the blood concentration of

hemoglobin, and SaO2 is the arterial oxygen saturation.

The venous oxygen content (CvO2) was computed as:

[

₂*0.003

]

* ₂ *1.39

2 PvO Hb SvO

CvO = +

where PvO2 is venous partial pressure of oxygen, Hb is the blood concentration of hemoglobin,

and SvO2 is the venous oxygen saturation.

As only 22 patients had a pulmonary artery catheter, while 63 had a central venous catheter, in the 41 patients having only a central venous catheter blood gas values obtained from the central venous blood were used for the computation of the right-to-left intrapulmonary shunt as a surrogate of mixed venous blood values.

4) The respiratory-system compliance (ml/cm of H2O):

PEEP pressure plateau V compliance system y respirator T − =

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where VT is the tidal volume, plateau pressure is the inspiratory plateau pressure, and PEEP is

the positive end-expiratory pressure. PEEP values were corrected for values of intrinsic PEEP, when detected during an end-expiratory pause [2].

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CT-scan image analysis

Approach to the quantitative CT-scan analysis

The CT scan measures the reduction of the radiation intensity upon passage through matter, which is called “linear attenuation coefficient” (µ) [3]. Through different mathematical algorithm, a given attenuation number µ is assigned to each voxel. The attenuation number depends on the energy of x-ray photons, as well as on density and the atomic number (Z) of material [4]. As a result, the attenuation is primarily determined by the density (mass/volume) of the tissue. In a given voxel the CT number is expressed as:

(

)

water water CT µ µ µ− =1000*

where CT is the mean CT number, µ is the linear attenuation coefficient of the given material, and µwater is the linear attenuation coefficient of water.

Indeed, for practical purposes the CT number is a measure of density [5], i.e.:

(

Volumeof gas Volumeof tissue

)

gas of Volume CT + = −1000

In the present paper, we refer to the tissue weight [6], i.e.:

volume Total CT weight Tissue * 1000 1 ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ − − =

We believe that referring to tissue instead of volume has several advantages, as what is subjected to stress and strain during mechanical ventilation is specifically the lung tissue, and not the lung volume, or the volume of lung regions with different degrees of aeration.

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CT-scan image processing

The procedure to perform the quantitative analysis of each single CT-scan image was performed for all the ALI/ARDS patients, as well as for all the patients with either healthy lungs or

unilateral pneumonia, at the Istituto di Anestesia e Rianimazione, Fondazione IRCCS – “Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena” di Milano. Each single CT-slice was considered as a region of interest. In each slice, the outlines of the lungs were manually countered, following the internal rib margin, the external mediastinal margin and the diaphragm profile. Pleural effusions, as well as large vessels of lung hilum, were excluded from the

countered image. Before quantitative analysis, the accuracy of each single countered images was assessed by a Radiologist (B.F., from the Department of Radiology, Fondazione IRCCS – “ Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena” di Milano), blinded as to the patient and airway pressure applied. Moreover, the repeatability of this procedure was tested in five patients: the difference in the quantitative analysis obtained was within 2 percent (data not shown)

In each CT slice, we first determined the frequency distribution of the CT numbers of a given lung regions [5]. Then, we arbitrarily defined four lung compartments corresponding to different degrees of aeration, using the most adopted CT thresholds in the literature [6]: non-aerated lung tissue (with a density between +100 HU and –100 HU), poorly-aerated lung tissue (with a density between –101 HU and –500 HU), normally-aerated lung tissue (with a density between – 501 HU and –900 HU), and hyper-inflated lung tissue (with a density between –901 HU and – 1000 HU). In mathematical terms, for a given compartment:

(

numberof voxels

)

volumeof voxel

t compartmen of

volume = _compartmen_t ∗

where “volume of compartment” is the volume of a specific compartment having a particular degree of aeration, “(number of voxels)compartment” is the number of voxels included in that

specific compartment (having that specific degree of aeration), and “volume of voxels” is the volume of a voxel.

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We believe important to understand the difference between the computation of lung

compartment volume and the computation of lung compartment tissue weight. It is evident that the tissue weight equals lung volume only in the non-aerated compartment, where the average CT is equal to zero. For the other compartments, the computation of tissue weight of a specific lung compartment is derived by using the average CT number of that specific compartment in the formula of the tissue weight computation.

Definition of the potentially recruitable lung

Among the possible different methods to compute lung recruitment, i.e. the recruitment to aeration of a portion of the lung parenchyma, we choose to define lung recruitment as the difference of the non-aerated lung tissue between 5 cm of H2O PEEP and 45 cm of H2O

inspiratory airway pressure, and expressed as a proportion of the total lung weight. We chose this method as the damages of mechanical ventilation to the lungs are primarily induced by the formation of intra-parenchymal shear forces, that are more likely to develop when a collapsed tissue becomes de-collapsed. From an anatomical point of view, these modifications within the lung parenchyma are reflected by the transformation of non-aerated lung tissue to aerated lung tissue. We believe that other methods to calculate lung recruitment (such as the volume of gas entering the poorly-aerated lung compartment [7]), are very likely to be correlated with gas exchange modifications, but less correlated with the physical triggers of the ventilator-induced lung injury.

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A

DDITIONAL RESULTS

Overall population and higher versus lower amount of recruitable lung tissue

Potentially recruitable lung and lung injury severity

The proportion of non-aerated lung tissue measured at 5 cm of H2O PEEP, an index of the

underlying severity of the lung injury, was widely variable and normally distributed (Figure 1 of the Supplementary Appendix), with a mean proportion equal to 37±16 percent of the total lung tissue (95 percent confidence interval, 33 to 41 percent; median 37 percent), corresponding to an absolute amount of 587±392 grams (95 percent confidence interval, 492 to 682 grams; median 476 grams).

The amount of potentially recruitable lung (PRL) appears to be a function of the proportion of non-aerated lung tissue to the total lung weight measured at the baseline 5 cm of H2O PEEP

(r2=0.46, P<0.001, Figure 2 of the Supplementary Appendix). Of note, the prevalence of patients that were dead at ICU discharge was greater among patients with either higher amount of PRL or higher proportion of non-aerated lung tissue at 5 cm of H2O PEEP, in comparison with that

observed among patients either with lower amount or PRL or lower proportion of non-aerated lung tissue at baseline PEEP (solid circles, Figure 2 of the Supplementary Appendix). Similarly, the survival during the first 28 days of ICU admission observed in patients with a higher amount of PRL was significantly shorter than that observed in patients with a lower amount of PRL (P=0.02, Figure 3 of the Supplementary Appendix).

PEEP trial and estimation of the potentially recruitable lung

Several physiological respiratory variables recorded at 5 and 15 cm of H2O of PEEP were

closely associated with the amount of PRL (Table 1 of the Supplementary Appendix). However, considering in particular their variation between 5 and 15 cm of H2O PEEP, the amount of PRL

was significantly associated only with the variation of arterial oxygen saturation (P<0.001), respiratory-system compliance (P=0.003), right-to-left intrapulmonary shunt fraction (P=0.03),

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venous partial pressure of oxygen (P=0.004), and venous oxygen saturation (P=0.001), a higher amount of PRL being associated with greater variations.

Table 2 of the Supplementary Appendix shows the sensitivity and specificity of tests set up by using combined physiological respiratory variables to predict patients with a higher amount of PRL, both as originally hypothesized and tested as post-hoc analysis.

Patients with ALI/ARDS from pneumonia and patients with ALI/ARDS from sepsis

As reported in the main text of the manuscript, patients with ALI/ARDS from pneumonia appeared to be more frequent among patients with a higher amount of PRL, while patients with ALI/ARDS from sepsis were more frequent among patients with a lower amount of PRL, in contrast with previous investigations [8,9]. To investigate for any possible explanation of these findings, we analyzed the baseline clinical characteristics and CT-scan variables of ALI/ARDS patients from either pneumonia or sepsis. Unexpectedly, patients with ALI/ARDS from

pneumonia showed a greater baseline severity of lung injury, as detected by a lower PaO2/FIO2

(P<0.001) and respiratory-system compliance (P=0.05), and a higher dead-space (P=0.02) and shunt fraction (P=0.001) than ALI/ARDS with sepsis (Table 3 of the Supplementary Appendix). Similarly, patients with ALI/ARDS from pneumonia showed a greater lung tissue weight

(P<0.001) and proportion of non-aerated lung tissue (P=0.04), and a lower proportion of

normally-aerated lung tissue (P<0.001) than patients with ALI/ARDS from sepsis (Table 4 of the Supplementary Appendix). Moreover, similar findings were observed when the analysis was limited to ALI/ARDS patients from pneumonia and ALI/ARDS patients from sepsis who underwent the CT-scan study protocol within 3 days from the diagnosis, thereby ruling out the possible effect of time on these results (data not shown). Therefore, it is very likely that

pneumonia led to a greater severity of lung injury as compared to sepsis syndrome in ALI/ARDS patients, and that, as a consequence, patients with ALI/ARDS from pneumonia had a greater amount of PRL. Indeed, these findings appear to follow the general message of the main manuscript: the greater the severity of lung injury, the greater is the amount of PRL.

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Patients with unilateral pneumonia

Thirty-four patients with unilateral pneumonia who underwent a whole lung CT-scan for

diagnostic purposes were retrospectively selected and included in the study as comparison group. Among this group, 20 patients were spontaneously breathing and 14 patients were mechanically ventilated. As expected, patients mechanically ventilated showed a greater severity of their systemic illness, as indicated by the SAPS II score (P=0.01) and the FIO2 clinically employed

(P=0.04), and higher values of PaCO2 (P=0.002), as compared to patients in spontaneous

breathing (Table 5 of the Supplementary Appendix). In contrast, the two groups of patients appeared to be greatly comparable with regard to the lung CT-scan functional anatomy, as judged by the lung tissue weight, the proportion of non-aerated lung tissue and the proportion of normally-aerated lung tissue (Table 5 of the Supplementary Appendix).

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Figure 1 – Supplementary Appendix.

The frequency distribution of non-aerated lung tissue recorded at 5 cm of H2O PEEP in the

overall study population (n=68), expressed as a proportion of the total lung weight. Dashed columns represent patients classified as affected by acute lung injury without ARDS (PaO2/FIO2

less than 300), while gray columns represent patients classified as affected by ARDS (PaO2/FIO2

less than 200). The non-aerated lung tissue was defined as the lung tissue having a physical density at CT-scan image analysis between +100 HU and –100 HU, representing the portion of lung parenchyma which is consolidated and/or collapsed, i.e. the lung injury severity.

non-aerated lung tissue [% total lung weight]

-10 / -5 -5 / 0 0 / 55 / 10₁₀ / 1₁₅5 / 2₂₀0 / 2₂₅5 / 3₃₀0 / 3₃₅5 / 4₄₀0 / 4₄₅5 / 5₅₀0 / 5₅₅5 / 6₆₀0 / 6₆₅5 / 7₇₀0 / 7₇₅5 / 80

fr

equenc

y

[

no.

of pat

ient

s

]

0 1 2 3 4 5 6 7 8 9 10 11

Figure 1 - Supplementary Appendix

ALI patients without ARDS ARDS patients

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Figure 2 – Supplementary Appendix.

The amount of PRL as a function of the severity of the lung injury, as estimated by the proportion of non-aerated lung tissue at 5 cm of H2O PEEP, in the overall study population

(n=68). Open circles represent patients surviving to discharge from the Intensive Care Units (ICUs, after 29±27 days), closed circles represent patients dying before ICU discharge. The dashed horizontal line represents the median value of the amount of PRL (9 percent of the total lung weight; 95 percent interval confidence, 8 to 14 percent). An exponential function (y = y0 +

a*exp(b*x)) was used to describe the relationship between the amount of PRL and the proportion of non-aerated lung tissue.

non-aerated lung tissue [% total lung weight]

0 10 20 30 40 50 60 70 80

the am

ount

of pot

ent

ially

recruit

able lung

[%

t

o

tal lung w

e

ight

]

-20 -10 0 10 20 30 40 50 60 70 80

dead survived

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The survival observed during the first 28 days of ICU admission in patients with either a lower or a higher amount of PRL. The dashed line represents patients with a lower amount of PRL (n=34), while solid lines represents patients with a higher amount of PRL (n=34). By the 28th day, 2 patients with a lower amount of PRL and 9 patients with a higher amount of PRL had died (6 vs. 26 percent, respectively, P=0.02).

days after admission to Intensive Care Unit

0 5 10 15 20 25 30

surv

iv

al [

%

]

0 20 40 60 80 100

lower amount of potentially recruitable lung higher amount of potentially recruitable lung

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Table 1 of the Supplementary Appendix – Physiological respiratory variables at different PEEP levels* Lower amount of PRL (≤ 9 percent) n = 34 Higher amount of PRL (> 9 percent) n = 34 P value†

Plateau pressure at 5 PEEP (cm of H2O) 18 ±3 21 ±4 <0.001

Plateau pressure at 15 PEEP (cm of H2O) 29 ±4 30 ±4 0.32

PaO2/FIO2 at 5 PEEP (mm Hg) 194 ±65 135 ±60 <0.001 PaO2/FIO2 at 15 PEEP (mm Hg) 245 ±88 202 ±73 0.03 Delta PaO2/FIO2 (mm Hg) 51 ±53 67 ±51 0.22 PaO2 at 5 PEEP (mm Hg) 87 ±22 71 ±22 0.002 PaO2 at 15 PEEP (mm Hg) 111 ±35 111 ±43 0.98 Delta PaO2 (mm Hg) 24 ±27 40 ±37 0.05 SaO2 at 5 PEEP (%) 96 ±3 92 ±5 <0.001 SaO2 at 15 PEEP (%) 97 ±2 97 ±2 0.74 Delta SaO2 (%) 1 ±2 5 ±4 <0.001 PaCO2 at 5 PEEP (mm Hg) 39 ±7 44 ±10 0.02 PaCO2 at 15 PEEP (mm Hg) 39 ±7 45 ±11 0.03 Delta PaCO2 (mm Hg) 0 ±3 1 ±4 0.70 Arterial pH at 5 PEEP 7.43 ±0.09 7.37 ±0.07 0.003 Arterial pH at 15 PEEP 7.42 ±0.09 7.35 ±0.07 0.001

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Dead space at 5 PEEP (% of tidal-volume)‡ 51 ±12 63 ±13 0.002

Dead space at 15 PEEP (% of tidal-volume)‡ 53 ±12 63 ±13 0.002

Delta dead space (% of tidal volume)‡ 2 ±5 0 ±4 0.25

Alveolar-dead space at 5 PEEP

(% of tidal volume)§ 15 ±11 25 ±13 0.002

(% of tidal volume)§ 15 ±12 23 ±13 0.02

Delta alveolar-dead space

(% of tidal volume)§ -0 ±7 -3 ±9 0.32

Respiratory-system compliance at 5 PEEP

(ml/cm of H2O)¶

51 ±19 38 ±15 0.002

Respiratory-system compliance at 15 PEEP

(ml/cm of H2O)¶

46 ±17 40 ±16 0.10

Delta respiratory-system compliance

(ml/cm of H2O)¶

-5 ±10 2 ±9 0.003

Shunt at 5 PEEP (% of cardiac output)|| 34 ±12 45 ±17 0.008

Shunt at 15 PEEP (% of cardiac output)|| 28 ±10 35 ±14 0.09

Delta shunt (% of cardiac output)|| -6 ±6 -10 ±8 0.03

PvO2 at 5 PEEP (mm Hg) 41 ±5 41 ±6 0.92

PvO2 at 15 PEEP (mm Hg) 42 ± 7 45 ±8 0.12

Delta PvO2 (mm Hg) 1 ±4 4 ±5 0.004

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SvO2 at 15 PEEP (%) 76 ±8 78 ±7 0.44 Delta SvO2 (%) 0 ±4 5 ±5 0.001 PvCO2 at 5 PEEP (mm Hg) 44 ±7 48 ±9 0.07 PvCO2 at 15 PEEP (mm Hg) 45 ±7 49 ±9 0.12 Delta PvCO2 (mm Hg) 1 ±4 1 ±4 0.59 Venous pH at 5 PEEP 7.38 ±0.08 7.34 ±0.07 0.03 Venous pH at 15 PEEP 7.38 ±0.08 7.33 ±0.07 0.02

* Values are mean ±SD. PEEP denotes positive end-expiratory pressure values, PaO2 the arterial partial pressure of oxygen, FIO2 the

inspired oxygen fraction, SaO2 the arterial oxygen saturation, PaCO2 the arterial partial pressure of carbon dioxide, PvO2 the venous

partial pressure of oxygen, SvO2 the venous oxygen saturation, and PvCO2 denotes the venous partial pressure of carbon dioxide. The

delta of each variable was calculated as the difference between the values recorded at 15 cm of H2O PEEP minus the values recorded

at 5 cm of H2O PEEP.

† P values were obtained by Student’s t-test, and Wilcoxon’s test analysis as appropriate.

‡ The dead space was calculated using a standard formula (see above). This measurement was available for 48 patients (23 patients with a lower, and 25 patients with a higher amount of PRL).

§ The alveolar-dead space was calculated using a standard formula (see above). This measurement was available for 55 patients (27 patients with a lower, and 28 patients with a higher amount of PRL).

¶ Respiratory-system compliance was calculated as the ratio between the tidal volume and the difference between inspiratory plateau pressure and PEEP.

|| The intrapulmonary right-to-left shunt was calculated using a standard formula (see above). This measurement was available for 60 patients (29 patients with a lower, and 31 patients with a higher amount of PRL).

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Table 2 of the Supplementary Appendix – Sensibility, specificity, positive and negative predictive values of different tests to

estimate patients with a higher amount of potentially recruitable lung*

Sensitivity % (no. of patients) Specificity % (no. of patients) Positive predictive value % Negative predictive value %

Delta PaO2/FIO2 > 0 mm Hg, delta PaCO2 < 0 mm Hg,

delta respiratory-system compliance > 0 ml/cm of H2O†§ 71 (24/34) 59 (20/34) 63 67

Delta PaO2/FIO2 > 0 mm Hg,

delta alveolar-dead space < 0 % of tidal-volume‡,

delta respiratory-system compliance > 0 ml/cm of H2O†§

86 (24/28) 52 (14/27) 65 78

PaO2/FIO2 at 5 PEEP < 150 mmHg 74 (25/34) 76 (26/34) 76 74

PaO2/FIO2 at 5 PEEP < 150 mmHg,

delta PaCO2 < 0 mm Hg,

59 (20/34) 82 (28/34) 77 67

PaO2/FIO2 at 5 PEEP < 150 mm Hg,

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* PaO2 denotes the arterial partial pressure of oxygen,FIO2 the inspired oxygen fraction, PaCO2 the arterial partial pressure of carbon

dioxide, and PEEP denotes positive-end expiratory pressure levels. The delta of each variable was calculated as the difference between value recorded at 15 cm of H2O PEEP minus the values recorded at 5 cm of H2O PEEP.

† Respiratory-system compliance was calculated as the ratio between the tidal volume and the difference between inspiratory plateau pressure and PEEP.

‡ The alveolar-dead space was calculated using a standard formula (see above). This measurement was available for 55 patients (27 patients with a lower, and 28 patients with a higher amount of PRL).

§ This test was considered positive when two or three of the variable variations indicated occurred when increasing PEEP from 5 to 15 cm of H2O.

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Table 3 of the Supplementary Appendix – Baseline clinical characteristics and mortality rate of patients with ALI/ARDS from

either pneumonia or sepsis at PEEP of 5 cm of H2O*

Overall ALI/ARDS

from pneumonia

ALI/ARDS

from sepsis P value†

n = 49 n = 25 n = 24

Age (yrs) 53 ±16 51 ±17 54 ±14 0.51

Female sex – no. of patients (%) 31 (63) 17 (68) 10 (42) 0.06

Body mass index (kilogram/meters2) 25 ±5 24 ±4 27 ±5 0.02

SAPS II‡ 36 ±12 35 ±12 37 ±12 0.77

Tidal-volume (ml) 529 ±103 500 ±88 559 ±110 0.04

Minute ventilation (liters/min) 10.2 ±2.8 10.5 ±3.1 9.8 ±2.6 0.39

Respiratory rate (breaths/min) 20 ±7 21 ±7 18 ±6 0.11

PEEP (cm of H2O) 11 ±3 11 ±3 11 ±3 0.96 PaO2/FIO2 (mm Hg) 163 ±72 130 ±52 198 ±75 <0.001 PaO2 (mm Hg) 78 ±23 71 ±19 85 ±25 0.06 FIO2 (%) 53 ±16 60 ±18 46 ±11 0.003 PaCO2 (mm Hg) 42 ±9 44 ±10 40 ±9 0.18 pH 7.39 ±0.09 7.38 ±0.09 7.40 ±0.08 0.33

Dead space (% of tidal-volume)§ 58 ±15 64 ±13 52 ±14 0.02

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Shunt (% of cardiac output)|| 40 ±17 48 ±18 33 ±12 0.001

Fluid balance before the study (ml/day)** 2477 ±3665 1496 ±3471 3457 ±3660 0.06

Days of ventilation before the study†† 6 ±6 5 ±6 6 ±6 0.45

Intra-abdominal pressure (cm of H2O) 13 ±5 11 ±4 16 ±5 0.001

Mortality at ICU discharge

(no. of patients, %)‡‡ 15 (31) 9 (36) 6 (25) 0.40

* Values are mean ±SD. PEEP denotes positive end-expiratory pressure values, FIO₂ the inspired oxygen fraction, PaO₂ the arterial

partial pressure of oxygen, and PaCO2 denotes the arterial partial pressure of carbon dioxide.† P values were obtained by Student’s

t-test, Wilcoxon’s t-test, and Chi-square test analysis as appropriate.

‡ The Simplified Acute Physiology Score II (SAPS II) [10] was used to assess the severity of systemic illness at study entry. Possible scores range from 0 to 163, with higher scores indicating more severe illness.

§ The dead space was calculated using a standard formula (see above). This measurement was available for 35 patients (19 patients with ALI/ARDS from pneumonia, and 16 patients with ALI/ARDS from sepsis).

¶ Respiratory-system compliance was calculated as the ratio between the tidal volume and the difference between inspiratory plateau pressure and PEEP.

|| The intrapulmonary right-to-left shunt was calculated using a standard formula (see above). This measurement was available for 43 patients (20 patients with ALI/ARDS from pneumonia, and 23 patients with ALI/ARDS from sepsis).

** Fluid balance before the study averaged for each patient the daily fluid balance within the last five days before the study. †† Days of mechanical ventilation before the study were counted from the day of intubation (day 0) to the day of the study. ‡‡ The average time of discharge from ICU was 29 ±27 days, ranging from 2 to 163 days (median 22.5 days).

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Table 4 of the Supplementary Appendix – Lung CT-scan variables of patients with ALI/ARDS from either pneumonia or

sepsis at PEEP of 5 cm of H2O*

Overall ALI/ARDS

from pneumonia

ALI/ARDS

from sepsis P value†

n = 49 n = 25 n = 24

Potentially recruitable lung (grams) 230 ±256 357 ±300 107 ±111 0.001

Potentially recruitable lung

(% of total lung weight) 13 ±13 19 ±14 8 ±7 0.002

Lung volume (ml) 2623 ±737 2784 ±836 2455 ±589 0.17

Lung tissue weight (grams) 1521 ±529 1780 ±551 1252 ±346 <0.001

Gas volume (ml) 1102 ±609 1004 ±706 1204 ±482 0.09

Non-aerated lung tissue (grams) 617 ±416 798 ±445 428 ±285 0.003

Non-aerated lung tissue

Consolidated lung tissue (grams) 387 ±265 451 ±285 321 ±230 0.09

Consolidated lung tissue

Poorly-aerated lung tissue (grams) 554 ±307 690 ±338 413 ±190 0.003

Poorly-aerated lung tissue

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Normally-aerated lung tissue (grams) 348 ±168 288 ±169 410 ±146 0.01

Normally-aerated lung tissue

(% of total lung weight) 26 ±16 18 ±13 35 ±14 <0.001

Hyper-inflated lung tissue (grams) 2 ±11 4 ±15 1 ±3 0.24

Hyper-inflated lung tissue

* Values are mean ±SD. Non-aerated lung tissue denotes the portion of lung parenchyma having density between +100 HU and –100 HU, consolidated lung tissue the portion of non-aerated lung tissue which remained non-aerated even at 45 cm of H2O airway

pressure, poorly-aerated lung tissue the portion of lung parenchyma having a density between –101 HU and –500 HU, normally-aerated lung tissue the portion of lung parenchyma having a density between –501 HU and –900 HU, and hyper-inflated lung tissue denotes the portion of lung parenchyma having a density between –901 HU and 1000 HU. The potential for lung recruitment was defined as the portion of non-aerated lung tissue regaining aeration from 5 to 45 cm of H2O airway pressure.

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Table 5 of the Supplementary Appendix – Clinical characteristics and baseline lung CT-scan variables in patients with

unilateral pneumonia during either spontaneous breathing or mechanical ventilation*

Patients with unilateral pneumonia spontaneously breathing n = 20 Patients with unilateral pneumonia mechanically ventilated n = 14 P value† Age 69 ±18 59 ±17 0.12 SAPS II‡ 28 ±8 46 ±20 0.01 PaO2/FIO2 (mm Hg) 235 ±102 200 ±104 0.93 FIO2 (%) 35 ±15 54 ±28 0.04 SaO2 (%) 93 ±4 89 ±7 0.28 PaCO2 (mm Hg) 36 ±6 46 ±7 0.002 Arterial pH 7.44 ±0.06 7.36 ±0.15 0.002 Lung volume (ml) 3531 ±1340 3099 ±795 0.31

Lung tissue weight (grams) 1150 ±335 1310 ±309 0.19

Gas volume (ml) 2382 ±1214 1790 ±653 0.11

Non-aerated lung tissue (grams) 291 ±219 449 ±253 0.04

Non-aerated lung tissue (% of total lung weight) 24 ±14 33 ±14 0.10

Poorly-aerated lung tissue (grams) 298 ±172 284 ±100 0.69

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Normally-aerated lung tissue (grams) 527 ±133 571 ±164 0.59

Normally-aerated lung tissue (% of total lung weight) 48 ±12 45 ±11 0.79

Hyper-inflated lung tissue (grams) 34 ±55 6 ±12 0.07

Hyper-inflated lung tissue (% of total lung weight) 3 ±4 1 ±1 0.06

Aerated-lung tissue (grams) 859 ±251 860 ±230 0.88

Aerated-lung tissue (% of total lung weight) 76 ±14 67 ±14 0.10

Mortality at hospital discharge (no. of patients, %)§ 4 (20) 2 (14) 1.0

* Values are mean ±SD. PaO2 denotes the arterial partial pressure of oxygen, FIO2 the inspired oxygen fraction, SaO2 the arterial

oxygen saturation, PaCO2 the arterial partial pressure of carbon dioxide, non-aerated lung tissue the portion of lung parenchyma

having a density between +100 HU and –100 HU, poorly-aerated lung tissue the portion of lung parenchyma having a density between –101 HU and –500 HU, normally-aerated lung tissue the portion of lung parenchyma having a density between –501 HU and –900 HU, and hyper-inflated lung tissue the portion of lung parenchyma having a density between –901 HU and 1000 HU, and aerated lung tissue denotes the portion of lung parenchyma having a density between –101 HU and –1000 HU, i.e. the sum of poorly-aerated, normally-aerated, and hyper-inflated lung tissue.

† P values were obtained Student’s t-test, Wilcoxon’s test, and Fisher exact test analysis as appropriate.

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Analysis according to the quartile distribution of the amount of potentially recruitable lung

To further characterize the distribution of the amount of PRL among our ALI/ARDS patient population, we divided the study population into for groups using the quartile values of the distribution of the amount of PRL: patients with a very low amount of PRL (less than 6 percent of the total lung weight), patients with a low amount of PRL (between 6 and 9 percent), patients with a high amount of PRL (between 9 and 19 percent), and patients with a very high amount of PRL (more than 19 percent of the total lung weight).

Clinical characteristics

The pre-study clinical characteristics of the four groups of patients were similar with regard to age, female sex prevalence, body mass index, severity of illness as assessed by the SAPS II score, daily fluid intake before the study, and days of mechanical ventilation before the study (Table 6 of the Supplementary Appendix). Tidal volume, PEEP and minute ventilation clinically employed were not different between the groups. In contrast, respiratory-system compliance and PaO2/FIO2 were progressively lower in patients with higher amounts of PRL in comparison with

patients with lower amount of PRL (P=0.009 and P=0.005, respectively, Table 6 of the

Supplementary Appendix). Moreover, the mortality rate, both 28 days after ICU admission and at ICU discharge, progressively increased along with the amount of PRL (P=0.002 and P=0.006, respectively, Table 6 of the Supplementary Appendix).

Bedside prediction of the amount of potentially recruitable lung

To provide an estimation at the bedside of the amount of PRL, patients underwent a PEEP trial at two different PEEP levels, i.e. 5 and 15 cm of H2O. As already described in the main text of the

manuscript, we initially hypothesized that patients with higher amounts of PRL would have shown at least two of the following respiratory variable responses when increasing PEEP from 5 to 15 cm of H2O: an increase in PaO2/FIO2, a decrease in PaCO2, and an increase in the

respiratory-system compliance. Indeed, this test succeeded in distinguish patients with higher amounts of PRL from patients with lower amounts of PRL with an acceptable accuracy, especially for the extreme quartiles (patients with a very low vs. patients with a very high amount of PRL). In contrast, the power of this test in characterizing the patients within the two

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intermediate quartiles (patients with a low and patients with a high amount of PRL) was quite poor (Table 7 of the Supplementary Appendix). Considering the variation of alveolar-dead space in place of the variation of PaCO2 increased the accuracy of detecting patients with a very high

amount of PRL (100 percent of the patients detected, Table 7 of the Supplementary Appendix), but did not improve the characterization and the distinction between the two intermediate quartiles. The use of the PaO2/FIO2 value at 5 cm of H2O PEEP in the place of its variation

between 5 and 15 cm of H2O PEEP significantly improve the accuracy of the estimation of the

amount of PRL. Patients with a PaO2/FIO2 value at 5 cm of H2O PEEP less than 150 mm Hg

were markedly more frequent within the two groups of patients with higher amounts of PRL as compared to the two groups of patients with lower amounts of PRL (Table 7 of the

Supplementary Appendix). Among all the combinations of physiological respiratory variables tested, the best predictor of patients with higher amounts of PRL appeared to be the presence of at least two of the following parameters: a PaO2/FIO2 value at 5 cm of H2O PEEP less than 150

mm Hg, a decrease in alveolar-dead space, and an increase in the respiratory-system compliance when increasing PEEP from 5 to 15 cm of H2O.

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Table 6 of the Supplementary Appendix – Baseline clinical characteristics and mortality rate of the study population

according to the quartile distribution of the amount of potentially recruitable lung*

Very low amount of PRL (≤ 6 percent) n = 17 Low amount of PRL (> 6 and ≤ 9 percent) n = 17 High amount of PRL (> 9 and ≤ 19 percent) n = 17 Very high amount of PRL (> 19 percent) n = 17 P value† Age (yrs) 57 ±17 55 ±16 60 ±18 47 ±16 0.12

Female sex – no. of patients (%) 8 (47) 7 (41) 4 (24) 14 (83) 0.11

Body mass index (kilogram/meters2) 26 ±5 25 ±4 26 ±5 23 ±3 0.33

SAPS II|| 38 ±11 35 ±13 34 ±8 39 ±10 0.41

Tidal-volume (ml/kg ideal body weight) 8.5 ±2.3 9.3 ±1.7 8.6 ±2.0 8.9 ±1.4 0.58

Minute ventilation (liters/min) 9.8 ±3.0 9.2 ±2.5 9.5 ±2.9 10.6 ±3.6 0.59

Respiratory rate (breaths/min) 19 ±7 16 ±5 17 ±6 21 ±7 0.15

PEEP (cm of H2O) 10.6 ±3.1 10.9 ±2.7 11.3 ±2.4 11.7 ±3.8 0.62 Plateau pressure (cm of H2O) 23 ±2 24 ±3 25 ±5 27 ±4‡§ 0.01 Respiratory-system compliance (ml/cm of H2O)** 50 ±15 48 ±16 46 ±21 33 ±11‡§ 0.009 PaO2/FIO2 (mm Hg) 231 ±53 218 ±84 204 ±76 148 ±71‡§ 0.005 FIO2 (%) 43 ±8 50 ±11 45 ±8 63 ±20‡§¶ 0.002

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PaCO2 (mm Hg) 38 ±7 38 ±10 42 ±11 49 ±21 0.07

Arterial pH 7.43 ±0.08 7.41 ±0.09 7.38 ±0.05 7.37 ±0.09 0.08

Causes of lung injury (no. of patients, %):

Pneumonia 3 (18) 4 (24) 6 (35) 12 (71) 0.007

Sepsis 10 (59) 7 (41) 5 (29) 2 (12) 0.03

Aspiration 2 (12) 1 (6) 1 (6) 0 (0) 0.90

Trauma 1 (6) 2 (12) 0 (0) 0 (0) 0.61

Others†† 1 (6) 3 (18) 5 (29) 3 (18) 0.40

Fluid balance before the study (ml/day)‡‡ 1304 ±2001 1550 ±2084 1356 ±2586 1440 ±1474 0.91

Days of ventilation before the study§§ 5 ±5 6 ±7 6 ±7 6 ±6 0.73

ALI / ARDS (no. of patients) 9 / 8 5 / 12 5 / 12 0 / 17 0.001

Mortality 28-days after ICU entry

(no. of patients, %) 0 (0) 2 (12) 2 (12) 7 (41) 0.002

(no. of patients, %)¶¶ 2 (12) 3 (18) 5 (29) 9 (53) 0.006

* Values are mean ±SD. PEEP denotes positive end-expiratory pressure values, PaO2 the arterial partial pressure of oxygen, FIO2 the

inspired oxygen fraction, PaCO2 the arterial partial pressure of carbon dioxide, and ICU denotes Intensive Care Unit. Because of

rounding, percentages may not total 100.

† P values were obtained by Student’s t-test, Fisher exact test, Chi-square test and Mantel-Haenszel’s test analysis as appropriate. ‡ P<0.01 vs. patients with a very low amount of PRL.

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¶ P<0.05 vs. patients with a high amount of PRL.

|| The Simplified Acute Physiology Score II (SAPS II) [10] was used to assess the severity of systemic illness at study entry. Possible scores range from 0 to 163, with higher scores indicating more severe illness.

** Respiratory-system compliance was calculated as the ratio between the tidal volume and the difference between inspiratory plateau pressure and PEEP.

†† Other causes of acute lung injury included anaphylactic shock, acute lung injury after surgery and following bone marrow transplantation.

‡‡ Fluid balance before the study averaged for each patient the daily fluid intake within the last five days before the study. §§ Days of mechanical ventilation before the study were counted from the day of intubation (day 0) to the day of the study. ¶¶ The average time of discharge from ICU was 29±27 days, ranging from 2 to 163 days (median 22.5 days).

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Table 7 of the Supplementary Appendix – Prediction of the amount of potentially recruitable lung according to its quartile distribution* Very low amount of PRL (≤ 6 percent) n = 17 Low amount of PRL (> 6 and ≤ 9 percent) n = 17 High amount of PRL (> 9 and ≤ 19 percent) n = 17 Very high amount of PRL (> 19 percent) n = 17

No. of patients with 2 or 3 of the following positive parameters (%):

delta PaO2/FIO2 > 0 mm Hg, delta PaCO2 < 0 mm Hg,

delta respiratory-system compliance > 0 ml/cm of H2O†

5 (29) 9 (53) 10 (59) 14 (82)

delta PaO2/FIO2 > 0 mm Hg,

3 (25) 10 (67) 9 (69) 15 (100)

No. of patients with PaO2/FIO2 at 5 PEEP < 150 mm Hg (%) 3 (18) 5 (29) 9 (53) 16 (94)

PaO2/FIO2 at 5 PEEP < 150 mm Hg, delta PaCO2 < 0 mm Hg,

delta respiratory-system compliance > 0 ml/ cm of H2O†

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PaO2/FIO2 at 5 PEEP < 150 mm Hg,

1 (8) 4 (27) 8 (62) 14 (93)

* PaO2 denotes the arterial partial pressure of oxygen,FIO2 the inspired oxygen fraction, PaCO2 the arterial partial pressure of carbon

dioxide, and PEEP denotes positive-end expiratory pressure levels. The delta of each variable was calculated as the difference between the values recorded at 15 cm of H2O PEEP minus the values recorded at 5 cm of H2O PEEP.

† Respiratory-system compliance was calculated as the ratio between the tidal volume and the difference between inspiratory plateau pressure and PEEP.

‡ The alveolar-dead space was calculated using a standard formula (see above). This measurement was available for 55 patients (27 patients with a lower, and 28 patients with a higher amount of PRL).

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Predictors of mortality

To investigate any possible association between baseline respiratory and clinical variables and an increased risk of death in the overall study population, we first analyze the clinical, gas

exchange, and respiratory mechanics parameters recorded at baseline 5 cm of H2O PEEP

between patients who survived and patients who did not survive at discharge from ICU (29±27 days after ICU admission, Table 8 of the Supplementary Appendix), by using a univariate analysis (Student’s t-test or Wilcoxon’s test as appropriate). The variables significantly different between survivors and non-survivors were then grouped into two main categories: carbon dioxide-related variables (such as dead space, alveolar dead space, and CO2 production), and

other gas exchange-related variables (such as arterial pH, venous pH, and PvO2). For each

category, all the variables significantly different between survivors and non-survivors (P<0.05) were introduced into a stepwise, backward, multiple-logistic regression model. The SAPS II score and the heart rate at 5 cm of H2O PEEP were already considered for the subsequent

analysis. The respiratory and clinical variables identified in the two categories as independently associated with an increased risk of death, as well as the SAPS II score and heart rate at 5 cm of H2O PEEP, were re-introduced into a final stepwise, backward, multiple-logistic regression

model in order to identify among all the physiological respiratory and clinical variables the most independently associated with an increased risk of death.

Among the carbon dioxide-related variables significantly different from survivors and non-survivors at ICU discharge, only dead space at 5 cm of H2O PEEP appeared to be independently

associated with an increased risk of death (P=0.002). Among the other gas exchange-related variables, the only parameters independently associated with an increased risk of death was the arterial pH at 5 cm of H2O PEEP (P=0.01).

The following physiological respiratory and clinical variables were therefore introduced into a stepwise, backward, multiple-logistic regression model: SAPS II score, dead space at 5 cm of H2O PEEP, arterial pH at 5 cm of H2O PEEP, and heart rate at 5 cm of H2O PEEP. Among them,

the variables that appeared to be independently associated with an increased risk of death were SAPS II score (P=0.04), dead space at 5 cm of H2O PEEP (P=0.03), and heart rate at 5 cm of

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H2O PEEP (P=0.02, Table 9 of the Supplementary Appendix). The odds of death increased as

SAPS II score increased, dead space at 5 cm of H2O PEEP increased, and as heart rate at 5 cm of

H2O PEEP increased. The fit of the model was clearly good, as indicated by the Hosmer and

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Table 8 of the Supplementary Appendix – Baseline respiratory and clinical variables associated with an increased risk of death*

Survivors

n = 49

Non-survivors

n = 19 P value†

SAPS II 34 ±10 42 ±11 0.007

Dead space at 5 PEEP

(% of tidal-volume)‡ 53 ±12 67 ±12 <0.001

(% of tidal-volume)§ 18 ±13 25 ±12 0.05

Heart rate at 5 PEEP (beats/min) 86 ±19 106 ±17 <0.001

Arterial pH at 5 PEEP 7.41 ±0.08 7.35 ±0.08 0.002

Venous pH at 5 PEEP 7.38 ±0.07 7.32 ±0.08 0.007

PvO2 at 5 PEEP (mm Hg) 40 ±5 44 ±7 0.01

* Values are mean ±SD. PEEP denotes positive-end expiratory pressure values, and PvO2 the

venous partial pressure of oxygen.

† P values were obtained by Student’s t-test or Wilcoxon’s test analysis as appropriate.

‡ The dead space was calculated using a standard formula (see above). This measurement was available for 48 patients (23 patients with a lower, and 25 patients with a higher amount of PRL). § The alveolar-dead space was calculated using standard formula (see above). This measurement was available for 55 patients (27 patients with a lower, and 28 patients with a higher amount of PRL).

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Table 9 of the Supplementary Appendix – Odds ratios for the respiratory and clinical variables independently associated with an increased risk of death*

Odds ratio (95% CI) P value

SAPS II (per 1-point increase) 1.12 (1.01–1.25) 0.04

Dead space at 5 PEEP (per increase of 5 percent)† 1.51 (1.05–2.18) 0.03

Heart rate at 5 PEEP (per increase of 5 beats/min) 1.40 (1.05–1.86) 0.02

* Results were calculated by using a stepwise, backward, multiple-logistic regression. The odds of death increased as SAPS II score increased, the dead space at 5 cm of H2O PEEP increased,

and the heart rate at 5 cm of H2O PEEP increased (Hosmer and Lemeshow goodness-of-fit test,

P=0.9577, c=0.921). CI denotes confidence interval, SAPS II the Simplified Acute Physiology Score II and PEEP denotes positive end-expiratory pressure values.

† The dead space was calculated using a standard formula (see above). This measurement was available for 48 patients (23 patients with a lower, and 25 patients with a higher amount of PRL).

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Association between the proportion of consolidated lung tissue and clinical and physiological respiratory variables

To investigate any possible association between the amount of consolidated lung tissue, i.e. the proportion of lung tissue which remained non-aerated even at 45 cm of H2O airway pressure, and

the amount of PRL, as well as clinical and physiological respiratory variables, the overall study population was divided into patients with a lower and patients with a higher proportion of consolidated lung tissue (n=34 for each group), using the median value of its frequency distribution in the entire study population (26 percent of the total lung weight – 95 percent confidence interval, 20 to 30 percent –, corresponding to an absolute amount of 323 grams – 95 percent confidence interval, 243 grams to 422 grams; see Figure 5 of the Supplementary

Appendix).

Overall, the frequency distribution of the consolidated lung tissue appeared to be variable among the study population, ranging from 2 percent to 48 percent of the total lung tissue weight (36 grams to 1112 grams, respectively; see Figure 4 of the Supplementary Appendix). Of note, patients classified as affected by ALI without ARDS or by ARDS were widely and equally distributed along the entire distribution of the consolidated lung tissue, and no difference was observed with regard to the percentage of ALI/ARDS patients between patients with either a lower or a higher proportion of consolidated lung tissue (Table 10 of the Supplementary

Appendix). The pre-study clinical characteristics of the two groups of patients were similar with regard to age, female sex prevalence, body mass index, SAPS II score, daily fluid intake before the study, and days of mechanical ventilation before the study (Table 10 of the Supplementary Appendix). The ventilatory setting clinically employed, as well as the mechanics of the

respiratory system clinically recorded, were comparable between the two groups of patients. Similarly, no difference was observed between patients with a lower and patients with a higher proportion of consolidated lung tissue with regard to clinical arterial blood gas values and causes of lung injury. Of note, the two groups of patients showed also a similar mortality rate 28 days after ICU admission and at ICU discharge (15 vs. 18 percent, P=0.74, and 21 vs. 35 percent, P=0.18, respectively; Table 10 of the Supplementary Appendix).

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PEEP trial

None of the physiological respiratory variables recorded at 5 cm of H2O PEEP appeared to be

associated with the proportion of the consolidated lung tissue: all the gas exchange and

respiratory mechanics parameters were comparable between patients with a lower and patients with a higher proportion of consolidated lung tissue (data not shown). In contrast, at 15 cm of H2O PEEP, patients with a higher-proportion of consolidated lung tissue showed lower levels of

PaO2/FIO2 (196±74 vs. 252±84 mm Hg, P=0.009), PaO2 (99±29 vs. 123±44 mm Hg, P=0.01)

and respiratory-system compliance (38±14 vs. 48±17 ml/cm of H2O, P=0.02), and higher values

of alveolar-dead space (23±12 vs. 15±13 percent of tidal-volume, P=0.02) in comparison with those observed in patients with a lower proportion of consolidated lung tissue. Considering in particular the variations of the physiological respiratory variables when increasing PEEP, the proportion of consolidated lung tissue was significantly associated only with the variations of PaO2/FIO2, a higher proportion of consolidated lung tissue being associated with smaller

variations (45±45 vs. 74±56 mm Hg, P=0.04).

Differences in the morphological appearance of lung parenchyma as detected by CT-scan in association with the proportion of consolidated lung tissue was then analyzed at 5, 15 and 45 cm of H2O airway pressure. Patients with a lower proportion of consolidated lung tissue showed a

similar amount of PRL as compared to that detected in patients with a higher proportion of consolidated lung tissue (12±11 vs. 13±12 percent of the total lung tissue, respectively, P=0.69, corresponding to an absolute amount of 197±237 vs. 237±228, respectively, P=0.46). The proportion of non-aerated lung tissue measured at 5 and 15 cm of H2O PEEP was significantly

associated with the proportion of consolidated lung tissue, a higher proportion of non-aerated lung tissue being associated with a higher proportion of consolidated lung tissue (P<0.001 for both). In contrast, the two groups of patients showed a similar variation in non-aerated lung tissue when changing PEEP from 5 to 15 cm of H2O (-8±8 vs. -7±6 percent, P=0.58,

respectively). Finally, patients with a lower proportion of consolidated lung tissue showed a greater increase in normally-aerated lung tissue when increasing PEEP in comparison with that observed in patients with a higher proportion of consolidated lung tissue (13±6 vs. 7±5 percent, P<0.001).

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The frequency distribution of consolidated lung tissue, i.e. the amount of lung tissue which remained non-aerated even at 45 cm of H2O airway pressure, in the overall study population

(n=68), expressed as a proportion of the total lung weight. Dashed columns represent patients classified as affected by acute lung injury without ARDS (PaO2/FIO2 less than 300 mm Hg),

while gray columns represent patients classified as affected by ARDS (PaO2/FIO2 less than 200

mm Hg). The non-aerated lung tissue was defined as the lung tissue having a physical density at CT-scan image analysis between +100 HU and –100 HU, representing the portion of lung parenchyma which is consolidated and/or collapsed.

consolidated lung tissue [% total lung weight]

-10 / -5 -5 / 0 0 / 55 / 10₁₀ / 1₁₅5 / 2₂₀0 / 2₂₅5 / 3₃₀0 / 3₃₅5 / 4₄₀0 / 4₄₅5 / 5₅₀0 / 5₅₅5 / 6₆₀0 / 6₆₅5 / 7₇₀0 / 7₇₅5 / 80

frequency

[no. of patients]

0 1 2 3 4 5 6 7 8 9 10 11 12 13

ALI patients without ARDS ARDS patients

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Table 10 of the Supplementary Appendix – Baseline clinical characteristics and mortality rate of the study population in

association with the proportion of consolidated lung tissue*

Lower proportion of consolidated lung tissue

(≤ 26 percent)

n = 34

Higher proportion of consolidated lung tissue

(> 26 percent)

n = 34

P value†

Age (yrs) 57 ±15 53 ±19 0.36

Female sex – no. of patients (%) 17 (50) 16 (47) 0.81

Body mass index (kg/meters2) 26 ±5 24 ±4 0.15

SAPS II‡ 36 ±10 37 ±12 0.76

Tidal-volume (ml/kg ideal body weight) 8.9 ±2.0 8.7 ±1.8 0.73

Minute ventilation (liters/min) 9.9 ±3.0 9.7 ±3.1 0.87

Respiratory rate (breaths/min) 18 ±7 19 ±7 0.54

PEEP (cm of H2O) 11.0 ±2.8 11.3 ±3.3 0.93 Plateau pressure (cm of H2O) 25 ±4 25 ±4 0.54 Respiratory-system compliance (ml/cm of H2O)§ 45 ±18 43 ±16 0.69 PaO2/FIO2 (mm Hg) 217 ±87 184 ±63 0.22 FIO2 (%) 49 ±13 51 ±17 0.95

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PaCO2 (mm Hg) 43 ±17 41 ±10 0.92

Arterial pH 7.40 ±0.07 7.39 ±0.08 0.39

Pneumonia 11 (32) 14 (41) 0.62

Sepsis 13 (38) 11 (32) 0.80

Aspiration 1 (3) 3 (9) 0.61

Trauma 2 (6) 1 (3) 1.00

Others¶ 7 (21) 5 (15) 0.75

Fluid balance before the study (ml/day)|| 1304 ±2352 1514 ±1695 0.22

Days of ventilation before the study** 5.2 ±6.1 5.9 ±6.0 0.43

ALI / ARDS (no. of patients) 12 / 22 7 /27 0.18

(no. of patients, %) 5 (15) 6 (18) 0.74

(no. of patients, %)†† 7 (21) 12 (35) 0.18

* Values are mean ±SD. PEEP denotes positive end-expiratory pressure values, PaO2 the arterial partial pressure of oxygen,FIO2 the

inspired oxygen fraction, PaCO2 the arterial partial pressure of carbon dioxide, and ICU denotes Intensive Care Unit. Because of

rounding, percentages may not total 100.

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§ Respiratory-system compliance was calculated as the ratio between the tidal volume and the difference between inspiratory plateau pressure and PEEP.

¶ Other causes of acute lung injury included anaphylactic shock, acute lung injury after surgery and following bone marrow transplantation.

|| Fluid balance before the study averaged for each patient the daily fluid intake within the last five days before the study. ** Days of mechanical ventilation before the study were counted from the day of intubation (day 0) to the day of the study. †† The average time of discharge from ICU was 29±27 days, ranging from 2 to 163 days (median 22.5 days).

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Association between time-period of mechanical ventilation before the study and clinical and physiological respiratory variables

As the time elapsed between beginning of mechanical ventilation and the day of the study was highly variable among the study population, we thought to exclude any possible influence of such a time-period on the results observed, by analyzing the overall study population for possible associations between the number of days of mechanical ventilation before the study and the clinical, respiratory and CT-scan variables recorded during the study. For this purpose, the study group was divided into patients with a shorter- and patients with a longer-time period of

mechanical ventilation before the study (n=34 for each group), using the median value of its frequency distribution in the entire study population (3.5 days; 95 percent confidence interval, 2 to 4).

The pre-study clinical characteristics of the two groups of patients were similar with regard to age, female sex prevalence, body mass index, SAPS II score, and daily fluid intake before the study (Table 11 of the Supplementary Appendix). The ventilatory setting clinically employed, i.e., tidal-volume, minute ventilation, respiratory rate and PEEP level, was comparable between patients with a shorter- and patients with a longer-period of mechanical ventilation before the study. In contrast, patients with a longer-period of mechanical ventilation before the study showed a lower respiratory-system compliance (P=0.02), and a slightly lower level ofFIO₂ clinically employed before the study (P=0.05), as compared to those observed in patients with a shorter-period of mechanical ventilation before the study (Table 11 of the Supplementary Appendix). No difference was observed between the two groups of patients with regard to the other clinical arterial blood gas values and with regard to the causes of lung injury. Of note, the mortality rate both 28 days after ICU admission and mortality rate at ICU discharge were clearly similar between patients with either a shorter- or a longer-period of mechanical ventilation before the study (21 vs. 12 percent, P=0.32, and 29 vs. 26 percent, P=0.79, respectively; Table 11 of the Supplementary Appendix).

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PEEP trial

None of the physiological respiratory variables recorded at 5 cm of H2O PEEP, as well as the

physiological respiratory variables recorded at 15 cm of H2O PEEP, appeared to be associated

with the time-period between the intubation and the day of the study: all the gas exchange and respiratory mechanics parameters were comparable between patients with a shorter- and patients with a longer-period of mechanical ventilation before the study (data not shown). Considering in particular the variation of the physiological respiratory variables between 5 to 15 cm of H2O

PEEP, only the variation in PvCO2 was slightly associated with the numbers of days of

mechanical ventilation before the study, a longer-period of mechanical ventilation being associated with a greater variation (2±4 vs. 0±3 mm Hg, P=0.05).

Differences in the morphological appearance of lung parenchyma as detected by CT-scan in association with time-period of mechanical ventilation between intubation and the day of the study was then analyzed at 5, 15 and 45 cm of H2O airway pressure. None of the CT-scan

variables recorded at 5 and 15 cm of H2O PEEP, as well as their variation when increasing

PEEP, were different between patients with a shorter- and patients with a longer-period of mechanical ventilation before the study (data not shown). Of note, the two groups of patients were also similar with regard to the amount of PRL (13±12 vs. 12±10 percent of the total lung tissue, respectively, P=0.80), demonstrating that in our study population the amount of PRL was not affected by the time-period of mechanical ventilation between the intubation and the day of the study.

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Table 11 of the Supplementary Appendix – Baseline clinical characteristics and mortality rate of the study population in

association with the time between the day of intubation and the day of the study*

Shorter-time of mechanical ventilation

between intubation and study day

(≤ 3.5 days)

n = 34

Longer-time of mechanical ventilation

between intubation and study day

(> 3.5 days)

n = 34

P value†

Age (yrs) 55 ±17 55 ±17 0.98

Female sex – no. of patients (%) 13 (38) 20 (59) 0.09

Body mass index (kg/meters2) 25 ±4 25 ±5 0.66

SAPS II‡ 37 ±10 36 ±12 0.57

Tidal-volume (ml/kg ideal body weight) 8.6 ±2.0 9.1 ±1.8 0.68

Minute ventilation (liters/min) 9.5 ±2.6 10.0 ±3.4 0.71

Respiratory rate (breaths/min) 17 ±6 19 ±7 0.19

PEEP (cm of H2O) 12 ±3 11 ±3 0.70

Plateau pressure (cm of H2O) 24 ±4 26 ±4 0.04

Respiratory-system compliance

(ml/cm of H2O)§

(48)

PaO2/FIO2 (mm Hg) 186 ±74 214 ±78 0.21

FIO2 (%) 53 ±15 48 ±15 0.05

PaCO2 (mm Hg) 42 ±16 42 ±11 0.92

Arterial pH 7.39 ±0.08 7.40 ±0.08 0.71

Pneumonia 13 (38) 12 (35) 1.00

Sepsis 10 (29) 14 (41) 0.45

Aspiration 2 (6) 2 (6) 1.00

Trauma 1 (3) 2 (6) 1.00

Others¶ 8 (24) 4 (12) 0.34

Fluid balance before the study|| 1721 ±2361 1105 ±1605 0.39

Days of ventilation before the study** 2 ±1 9 ±7 <0.001

ALI / ARDS (no. of patients) 10 / 24 9 / 25 0.79

(no. of patients, %) 7 (21) 4 (12) 0.32

Mortality at ICU discharge (no. of patients, %)†† 10 (29) 9 (26) 0.79

* Values are mean ±SD. PEEP denotes positive end-expiratory pressure values, PaO2 the arterial partial pressure of oxygen,FIO2 the

inspired oxygen fraction, and PaCO2 denotes the arterial partial pressure of carbon dioxide. Because of rounding, percentages may not

total 100.

(49)

§ Respiratory-system compliance was calculated as the ratio between the tidal volume and the difference between inspiratory plateau pressure and PEEP.

¶ Other causes of acute lung injury included anaphylactic shock, acute lung injury after surgery and following bone marrow transplantation.

|| Fluid balance before the study averaged for each patient the daily fluid intake within the last five days before the study. ** Days of mechanical ventilation before the study were counted from the day of intubation (day 0) to the day of the study. †† The average time of discharge from ICU was 29±27 days, ranging from 2 to 163 days (median 22.5 days).