Study design
A total of 44 patients were enrolled in a prospective study that took place over the period May-November 2005. The study population was different than the one described recently [4]. Patients were hospitalized in the Department of Critical Care of the "Evangelismos" General Hospital and in the 2nd Department of Critical Care of the "ATTIKON" University Hospital of Athens. The study was approved by the Ethics Committee of both hospitals. All enrolled patients were intubated for at least 48 hours prior enrolment and they were aged above 18 years. Written informed consent was provided by their relatives.
Exclusion criteria were:
• any hematologic or solid tumor malignancy;
• any administration of G-CSF or CM-CSF over the last three months;
• neutropenia (≤500 neutrophils/mm3);
• HIV infection; and
• oral intake of corticosteroids at a dose equal to or higher than 1 mg/kg equivalent of prednisone for a period greater than one month.
Inclusion criteria were the concomitant presence of:
• ventilator-associated pneumonia (VAP), and
• sepsis, severe sepsis or septic shock.
Death due to sepsis was the clinical endpoint of the study.
Definitions
Diagnosis of VAP was established in any patient presenting with the following signs:
• core temperature >38°C or <36°C;
• new or persistent consolidation in lung X-ray;
• purulent trancheobronchial secretions (TBS);
• clinical pulmonary infection score (CPIS) more than 6 [6–9].
CPIS was determined after individual scoring for each of the following parameters [10], as follows:
• Core temperature 36.5–38.4°C: 0 points; 38.5–38.9°C: 1 point; ≤36°C or ≥39°C: 2 points
• White blood cells 4,000–11,000/μl: 0 points; <4,000 or >11,000/μl: 1 point; >11,000 points and more than 10% bands: 2 points
• pO2/FiO2 ≥240 or presence of ARDS: 0 points; pO2/FiO2 <240 in the absence of ARDS: 2 points
• Diffuse shadows on lung X-ray: 1 point; localized shadow on lung X-ray: 2 points
• Purulent TBS: 2 points
• TBS cultures yielding a pathogen ≥106 cfu/ml with negative Gram stain: 1 point; TBS cultures yielding a pathogen ≥106 cfu/ml with positive Gram stain: 2 points
Diagnosis of sepsis was based on the presence of at least two of the following [11]:
• core temperature >38°C or <36°C;
• Pco2<32 mmHg;
• pulse rate >90/min; and
• white blood cells >12,000/μl or <4,000/μl or >10% bands.
Severe sepsis was determined as the acute dysfunction of at least one organ i.e. the acute presentation of at least one of the following [11]:
• Acute Respiratory Distress Syndrome (ARDS), as any value of pO2/FiO2 below 200
• Acute renal failure, as the production of less than 0.5 ml/Kg body weight/h of urine for at least two hours provided that the negative fluid balance of the patient was corrected
• Metabolic acidosis as any pH<7.30 or any base deficit greater than 5 mEq/l and serum lactate at least more than 2× normal value
• Acute coagulopathy as any platelet count <100.000/μl or INR> 1.5
Septic shock was considered as any value of systolic pressure below 90 mmHg requiring the administration of inotropic agents after adequate fluid resuscitation [11].
Sample collection
Upon enrolment in the study, quantitative cultures of tracheobronchial secretions (TBS) were performed; TBS were collected after insertion of a sterile catheter in the intubation tube or in the tracheotomy connected to a negative pressure device. Enrolled patients were followed-up on a daily basis for a total of 28 days; evaluation comprised lung X-rays, estimation of the pO2/FiO2 ratio and of the APACHE II and SOFA scores.
For the estimation of cytokines and of the percentage of CD34/CD45 cells, 5 ml of blood were sampled after venipuncture of a peripheral vein under sterile conditions on the first, third, fifth and seventh days. Blood was collected into sterile tubes for the estimation of cytokines and into EDTA-coated tubes (Vacutainer, Becton Dickinson, Cockeysville MD) for the determination of CD34/CD45 cells. After centrifugation, serum was kept at -70°C until assayed.
Laboratory techniques
Cultures of TBS
Quantitative TBS cultures were performed immediately after collection; 0.5 ml of sample were added into a sterile tube with 0.5 ml of 1 mg/ml of dithiothreitol (Oxoid Ltd, London, UK) and diluted five consecutive times 1:10. Volumes of 0.1 ml of each dilution were plated onto McConkey and blood agar (Becton Dickinson, Cockeysville, Md). Dishes were incubated for five days at 35°C and their count was estimated after multiplying with the appropriate dilution factor. Cultures yielding a pathogen at a count ≥1 × 106 cfu/ml were considered positive [10]. Identification of pathogens was performed by the API20E and the API20NE systems (bioMérieux, Paris L'Etoile, France).
Serum cytokine measurements
Concentrations of tumour necrosis factor-alpha (TNFα), interleukin-6 (IL-6), and IL-8 in sera were estimated in duplicate by an enzyme immunoabsorbent assay (Diaclone, Paris, France). Lowest limits of detection were 0.5 pg/ml for TNFα, 6.25 pg/ml for IL-6 and 62.5 pg/ml for IL-8. Concentrations of G-CSF (Granulocyte Colony Stimulating Factor) were estimated in sera of days 1 and 3 by an enzyme immunoabsorbent assay (R&D Systems, Minneapolis, USA). Lowest limit of detection was 1.25 pg/ml.
Flow cytometric analysis of hemopoetic cells
For the estimation of the percentage of CD34/CD45 cells, whole blood was applied. Red blood cells were lysed by the application of NH4Cl 0.1 M and white blood cells were washed three times with PBS pH: 7.2. Subsequently, they were incubated for 15 minutes in the dark with the monoclonal antibodies anti-CD45 FITC (Immuotech, Marseille, France) and anti-CD34 PE (Immunotech). Cells staining positive for both CD34 and CD45 after analysis by the EPICS XL/MSL flow cytometer (Beckman Coulter Co) with gating for the whole cell population, were considered hemopoetic stem cells [2]. IgG FITC and IgG PE antibodies were applied as negative controls for each sample. Mean fluorescence intensity (MFI) was also recorded. In order to evaluate the percentage of CD34/CD45 cells of the enrolled patient population, eight samples derived from healthy volunteers well matched for age and sex to the study population were applied. The absolute count of CD34/CD45 cells was estimated after multiplication of their percentage to the absolute white blood cell count determined after analysis by an automatic counter (Beckman Coulter Co).
Statistical analysis
Results were expressed as medians ± standard errors (SE) or 95% confidence intervals (CI). Comparisons between patients and healthy controls were performed by the Mann-Whitney U test. Statistical correlations were performed after assessment of the non-parametric co-efficient of Spearman (rs). Survival Kaplan-Meier analysis was performed separately for patients with CD34/CD45 cells less than 310/μl and equal to or more than 310/μl. The latter threshold was applied after scatterploting of single values of survivors and non-survivors. Survival curves were compared by the log-rank test. Cox regression analysis for estimation of hazard risk (HR) and their 95%CI for death due to sepsis was performed; APACHE II score of day 1 more than 20, category of critical illness and the count of CD34/CD45 cells on day 1 were covariates. Changes of CD34/CD45 counts were estimated between days 1 and 3, between days 1 and 5 and between days 1 and 7. These changes were compared for patients with CD34/CD45 cells less than 310/μl and equal to or more than 310/μl on day 1 by the Mann-Whitney U test. Any value of p below 0.05 was considered statistically significant.
