Bronchoalveolar lavage fluid characteristics and outcomes of invasively mechanically ventilated patients with COVID-19 pneumonia in Genoa, Italy

Background The primary objective of the study is to describe the cellular characteristics of bronchoalveolar lavage fluid (BALF) of COVID-19 patients requiring invasive mechanical ventilation; the secondary outcome is to describe BALF findings between survivors vs non-survivors. Materials and methods Patients positive for SARS-CoV-2 RT PCR, admitted to ICU between March and April 2020 were enrolled. At ICU admission, BALF were analyzed by flow cytometry. Univariate, multivariate and Spearman correlation analyses were performed. Results Sixty-four patients were enrolled, median age of 64 years (IQR 58–69). The majority cells in the BALF were neutrophils (70%, IQR 37.5–90.5) and macrophages (27%, IQR 7–49) while a minority were lymphocytes, 1%, TCD3+ 92% (IQR 82–95). The ICU mortality was 32.8%. Non-survivors had a significantly older age (p = 0.033) and peripheral lymphocytes (p = 0.012) were lower compared to the survivors. At multivariate analysis the percentage of macrophages in the BALF correlated with poor outcome (OR 1.336, CI95% 1.014–1.759, p = 0.039). Conclusions In critically ill patients, BALF cellularity is mainly composed of neutrophils and macrophages. The macrophages percentage in the BALF at ICU admittance correlated with higher ICU mortality. The lack of lymphocytes in BALF could partly explain a reduced anti-viral response.


Background
In December 2019, in China emerged a new coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the new disease caused by this virus is named coronavirus disease 2019 . The spectrum of clinical manifestations of SARS-CoV-2 infection is vast, ranging from asymptomatic or patients with few symptoms to complication of severe viral pneumonia with the acute respiratory distress syndrome (ARDS) [1][2][3][4]. An excessive inflammatory response to SARS-CoV-2 is a major cause of disease severity and death and is associated with high levels of circulating cytokines, severe lymphopenia and mononuclear cell lung infiltration [5]. There are two distinct but potentially overlapping pathological subsets [6], the first driven by the virus and the second by the host response. In the pulmonary disease, viral multiplication and inflammation in the lung is prevalent.
In addition, in lungs with characteristic diffuse alveolar damage [1] and in the bronchoalveolar lavage fluid (BALF) [7,8], monocytes and macrophages were prevalent, with a moderate numbers of multinucleated giant cells, and very few lymphocytes. Most of the infiltrating lymphocytes were CD4-positive T cells [1].
In the peripheral blood, a common feature in many patients with COVID-19, is the presence of a global T cell lymphopenia and this is particularly prominent in patients with more severe disease.
In patients infected by SARS-CoV-2, the lymphopenia of circulating T cells may be linked to their recruitment to inflamed tissues with a consequence of T cell depletion from the secondary lymphoid organs [9]. This finding is consistent with the "primary cytokine" storm induced by viral infection which is mainly produced by alveolar macrophages, epithelial and endothelial cells, rather than those in the "secondary cytokine" storm induced by various subsets of T lymphocytes in late stages of viral infection [10][11][12].
In the literature, there are few studies that analyze at same time both sites, peripheral blood and BALF cellularity and correlate these values with outcomes and clinical or immunological variables. In some studies that describe data only in one of two sites, the low percentage of patients had ARDS or critical pulmonary infection and the data are reported in few patients invasive mechanically ventilated [13,14].
The main aim of our study is to describe the BALF cellularity of patients admitted to the intensive care unit (ICU) and requiring invasive mechanical ventilation; the secondary outcome is to describe the BALF findings between survivors vs non survivors patients. As post-hoc analysis, we report the relationships with BALF data, clinical, immunological aspects and peripheral blood values which may predict prognosis.

Methods
Consecutive, critically ill patients requiring invasive mechanical ventilation for severe COVID-19 pneumonia, aged 18 years or over, admitted between March 5th and April 30th 2020 to the Intensive Care Unit (ICU) at San Martino University Hospital in Genova, Italy, were included in the present study. Confirmed infection was defined as real-time reverse-transcriptase polymerase chain reaction (RT-PCR) positive from a nasal and/or throat swab or BALF according to World Health Organization interim guidance [15] together with signs, symptoms and radiological findings suggestive of COVID-19 pneumonia. The study was carried out in accordance with the principles of the Declaration of Helsinki and approved by the Ethic Committee of Liguria Region (Comitato Etico Regione Liguria) (N. CER Liguria 114/2020 -ID 10420). Informed consent was waived by the Ethic Committee of Liguria Region (Comitato Etico Regione Liguria).

BALF collection
At admission to ICU, a first BALF was collected in each patient and the following were analyzed: total cellularity (%, 10 3 /ml), all cell subpopulations (lymphocytes, neutrophils, eosinophils, macrophages, monocytes), lymphocyte subtypes (T, B, NK), and T lymphocyte activation as HLA-DR expression.

Preparation of BALF
Fibroscopy was performed with patients sedated with propofol and midazolam and paralyzed with cisatracurium, otherwise intravenous boluses of midazolam or propofol were administered to provide sedation during flexible bronchoscopy with Ambu® aScope TM 4 Broncho Large 5.8 / 2.8. Chest radiography was performed within 3 h to guide the microbiological examination whereas right middle lobe or lingula were chosen in patients with bilateral pneumonia. We performed BAL by serial 20 ml fractions to a total volume of 100-120 ml of room temperature and 0.9% NaCl. BALF, about 60% of lavage volume, was retrieved by gentle syringe suction and put into sterile containers [16,17].

Flow cytometry of Broncho-alveolar lavage
The BALF sample containers were adequately disinfected before being sent to the laboratory. Once received, containers were collected in a ventilated room and the exterior of the containers re-disinfected. All sample handling was carried out by experienced staff who wore protective equipment including protective, disposable aprons, molded protection masks (FFP2), goggles and double layer gloves. Sample volume was noted including appearance, colour and possible contamination with peripheral blood. BALF samples were then filtered through a 70 μm nylon Cell Strainer filters (Thermo Fisher Scientific, Massachusetts, United States) and then centrifuged at 1500 rpm for 7 min. The resulting cell pellet was incubated with monoclonal antibodies (mAbs) in BD TruCount™ tubes for 15 min at room temperature in the dark, followed by the addition of FACS Lysing Solution for 15 min at room temperature. The working panel of mAbs at eight colour assays used for the lymphocyte and monocyte evaluation in BAL samples were the following: CD3 FITC/HLA-DR PE/CD4 PerCP-Cy5.5/CD56 PE-Cy7/CD19 APC/CD8 APCH7/CD15 H450/ CD45 VH500 (BD Biosciences, New Jersey, USA), CD66b FITC/HLA-DR PE/CD3 PerCP-Cy5.5/CD33 PE-Cy7/CD14 APC/CD16 APCH7/CD38 VH450/CD45 VH500 (BD Biosciences, New Jersey, USA). Once washed, samples were acquired within 1 h with a FACSCanto™ II flow cytometer (BD Biosciences, New Jersey, USA). The analysis of cytometric data on BALF samples was performed using BD FACSDiva™ software version 6.1.3 (BD Biosciences, New Jersey, USA). After acquisition and during analysis, the absolute number (cells/μL) of positive cells in the sample can be determined by comparing cellular events to bead events. BD FACSCanto™ clinical software (v2.0 or later) subsequently determines absolute counts.
The gating strategy used in this study is briefly described: debris were excluded on FSC-A and SSC-A plot, then, on FSC-A and FSC-W plot doublets were removed and all leucocyte cells (granulocytes, macrophagesmonocytes and lymphocytes) were identified by CD45+ vs SSC-A. The CD4+ and CD8+ T cells were further selected among CD3+ population. Before acquisition, instrument sensitivity was evaluated and monitored over time using the BDTM Cytometer Setting &Tracking system. In order to achieve consistent and comparable data over time, all the BD FACSCanto II cytometers were standardized using BD CS&T beads, creating an Application Setting and defining Target Values (TG). Before any acquisition, PMT voltages were updated using Application Setting and maintenance of TG were verified running beads [18,19].

Statistical analysis
For this descriptive observational study no sample size calculations were performed. In the descriptive analysis, categorical variables were summarized by means of numbers and percentages, whereas continuous variables were summarized through median values and interquartile ranges (IQR). Normal distribution variables were compared using the t-test and non-normal distribution continuous variables were compared with the Mann-Whitney test. Categorical variables were compared by the Chi-square test. The Pearson or Spearman correlation analysis was performed to show the correlation between clinical parameters and COVID-19 progression. To verify if there are some immunological aspects which may predict prognosis and distinguish between survivors and non survivors, demographic, clinical variables and laboratory values were tested for their association by means of univariate analysis. To assess the differences between groups, univariate logistic regression analysis with group of treatment as binary dependent variable was adopted. A multivariable regression model was made, with ICU-mortality as dependent variable. Variables were selected to be included in the multivariable model when a P < 0.10 was found in the univariable analysis. In addition, the following variables were chosen a priori to be included in the model because of their clinical relevance: age, sex, days from the onset of symptoms to ICU admission, interstitial pattern at chest radiography, PaO2/FiO2 ratio, TCD4+/TCD8+ ratio, neutrophils %, macrophages %. Odds ratios (OR) with 95% confidence intervals (CI) were reported. P value ≤0.05 was considered statistically significant. The analyses were performed using SPSS Statistics version 21.0 (IBM Corp., Armonk, NY, USA).
All parameters were collected in a relational database connected through a web-based interface for anonymous and automatic data collection [20]. The pathology laboratory database was used for these data collection.
In the BALF, the median value of macrophage percentages and activated lymphocytes (TCD3 + HLA-DR+) were higher in non-survivors compared to survivors (35% vs 20, and 23% vs 20%, respectively) while the TCD4+/TCD8+ ratio was lower in non-survivors compared to survivors (0.5 vs 0.6, respectively) ( Fig. 1Panel  A, B). All the differences are not statistically significant. The mean count of total cellularity in BALF was higher, but not significantly different, between survivors and non-survivors (250 vs 117, 10 3 /ml cells, respectively).

Discussion
In the present descriptive observational study, we found that BALF cellularity of mechanically ventilated patients with COVID-19 pneumonia was characterized mostly by neutrophils, macrophages and a minority of TCD3+ lymphocytes, with a majority of TCD8+, with low percentage of activation (HLA-DR+). In the BALF, the percentage of total cellularity and activated lymphocytes were higher while the TCD4+/TCD8+ ratio was lower in survivors compared to non-survivors. At post-hoc analysis, the percentage of macrophages in the BALF at ICU admittance correlated with higher ICU mortality.
The neutrophilic cellular pattern is typical of ARDS and diffuse alveolar damage (DAD) and as neutrophils release chemokines and cytokines this may explain the generation of the cytokine storm, which is a leading cause of death in patients with severe acute respiratory syndrome [12,13]. In this phase, recruitment of monocytes contributes to the rapid decline of alveolar patency and promotes ARDS [15].
As reported in recent literature [7], pulmonary involvement of SARS-CoV-2 starts in the second stage with viral multiplication and inflammation in the lungs with lymphopenia in peripheral blood.
There are two possible reasons for the reduction of T cells in patients with COVID-19, lymphocytes are either directly invaded by the virus or they are indirectly damaged by the induced cytokine storm [1].
In our cohort, BALF were collected at a median of 4 days from diagnosis of SARS-CoV-2, at a median of 9 days from the onset of symptoms and for all patients at the day of ICU admission. Therefore, these patients are probably within the second phase of viral infection and we are probably describing, from 9 to 14 days, the overlapping of the viral response phase and the host response inflammatory phase (6).
The peripheral CD8+ T cells lymphopenia in patients admitted to ICU correlates with COVID-19 severity and  [21,22]; TCD4+/TCD8+ ratio is elevated in the peripheral blood compared to BALF in this cohort of patients.
As reported in Table 3 [7, 8, 12-14, 21, 23, 24] there are few data concerning the description of BALF cellularity and peripheral blood in patients with critical pulmonary infection in COVID 19. In one study the analysis of BALF from COVID-19 patients revealed an increase in CD8 T cell infiltrate with clonal expansion [7]. In another study, post-mortem examination of a patient with ARDS in COVID-19 showed lymphocyte infiltration in the lungs [14]. Another study that analyzed post-mortem biopsies from four COVID-19 patients describes that in three patients in lung biopsy were found mononuclear infiltration [8] and in another one recently published, the inflammatory infiltrate, observed in all 38 cases, was largely composed of macrophages in the alveolar spaces (in 24 cases) and lymphocytes in the interstitium (in 31 cases) [24].
The peculiarity of our study is the analysis of cellularity both in BALF and in the peripheral blood of mechanically ventilated COVID-19 patients; we describe a reduction of TCD3+ and TCD8+ lymphocytes in BALF In the bronchoalveolar lavage fluid of non-survivors, the median value of macrophage percentages was higher (35%) than in survivors (20%); the TCD4+/TCD8+ ratio was lower (0.5 vs 0.6), and activated lymphocytes (TCD3 + HLA-DR+) were higher in the non-survivors (23% vs 20%) compared to survivors. All the differences are not statistically significant with a consequent decrease of TCD4/TCD8 ratio; we found that the majority of cells were neutrophils and macrophages. However, in peripheral blood, we observed an increase of the TCD4+/TCD8+ ratio.
Moreover, cellularity distinguished patients in survivors and non survivors; indeed, we found a reduction of peripheral lymphocytes and absolute TCD8+ in the nonsurvivor group of patients.
As post-hoc analysis, we found that older age and peripheral lymphopenia, specially TCD8+, correlated with poor outcome. In addition, we found a positively correlation between the duration of symptoms to ICU admission and the peripheral IL-6 values, to strengthen the hypothesis that in this second phase of infection the role of blood immunological markers and BALF cellularity are the main authors.
Fatal cases show persistent and more severe lymphopenia compared with recovered patients, suggesting that a cellular immune deficiency state may be associated with poor prognosis [10]. T cells play a crucial role in viral infections: the TCD4+ cells provide B cell-help for antibody production while TCD8+ cells kill infected cells to reduce viral burden [25,26]. In our descriptive analysis, we found a reduction of overall lymphocytes, both TCD4+ and TCD8+ with a higher ratio, in the peripheral blood compared to BALF. A negative correlation between monocytes and lymphocytes TCD3+ and also between %TCD4+ and %NK cells in BALF confirm the predominant role of innate immunity cells in this phase of lung damage.
In our study, the percentage of TCD3+ in BALF was inversely correlated with LDH in the blood; this result could explain this phase of lung damage, expressed with increased value of lactate dehydrogenase and the reduction of all lymphocytes in the lung. LDH is an important parameter to measure lung damage and/or lung dysfunction. Indeed, in the CALL score one of the four considered parameters is LDH and this appears to be a simple and accurate model for the prediction of COVID-19 progression to severe cases [27,28]. This hypothesis is supported by the fact that the duration of mechanical ventilation is inversely correlated with percentage of TCD8+ but shows positive correlation with BALF TCD4+/TCD8+ ratio. Overall the present data suggest that in the second phase COVID-19 with lung viral infection in the lungs there is a relative lack of lymphocytes, in BALF that reflects lymphocyte depletion in the lung [7], with decreased immune responses to control virus replication.
This study has some limitations to be addressed. First, the design of the study is observational and descriptive; second the number of patients is relatively small; third, we did not evaluate CT scan images, but only the radiographic findings and their correlation to BALF data and fourth, we did not evaluate an evolution time for BALF.

Conclusion
In critically ill patients with COVID-19 pneumonia requiring invasive mechanical ventilation, BALF cellularity is mainly composed of neutrophils and macrophages, with a minority of TCD3+ lymphocytes. The percentage of macrophages in the BALF at ICU admittance correlated with higher ICU mortality. The lack of No data >Macrophages >Lymphocytes a ARDS Acute Respiratory Distress Syndrome, b IMV Invasive mechanical ventilation, c n.a. not available lymphocytes in BALF, in the second phase of viral infection, could partly explain a reduced anti-viral response. Further investigation is therefore advisable to gain a better understanding of BALF information to guide efforts aimed at reducing the fatality rate and at clarifying the future evolution of pulmonary follow up of COVID-19 survivors.