Mini Invasive Probe Based Confocal Laser Endomicroscopy of the Pleura for Malignancies Diagnosis
Conditions: Pleural Diseases, Pleural Neoplasms
Sponsor: University of Liege

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Mini invasive pleural probe based confocal laser endomicroscopy for malignant pleural 
effusion diagnosis 
 
 
Study Title: Mini invasive pleural probe based confocal laser endomicroscopy for malignant 
pleural effusion diagnosis 
Study Sponsor: CHU de Liège Sart-Tilman 
Research Organization: Pneumology Department, CHU de Liège 
Medical Ethics Committee: University of Liège hospital-faculty ethics committee 
                              
Document date: 11/12/2020 
Local Investigator Physicians: 
Prof. B. Duysinx (Chief of Pneumology Clinic, CHU de Liège) 
Dr. V. Heinen (Chief of Pneumology Clinic, CHU de Liège) 
Dr. O. Bonhomme (Deputy Chief of Pneumology Clinic, CHU de Liège) 
 
 
NCT04731129 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Mini invasive pleural probe based confocal laser endomicroscopy for malignant pleural 
effusion diagnosis 
 
Study protocol  
 
Introduction 
 
Confocal laser microscopy was first introduced at the Princeton University by Professor Marvin 
Minsky. This optical imaging technique uses a pinhole at the confocal plane of a lens, selecting a 
very narrow parallel shaped beam coming from the confocal plane (located in the object of 
interest) and eliminating out of focus light. A laser is used as illuminating source of light, hence 
the technique name, because of its optical properties allowing better resolution. The LASER 
beam can then excite molecules in the confocal plane of the sample examined, resulting in 
autofluorescence of some molecules. The autofluorescent light emission can  then be captured 
and analyzed (1,2). In probe-based confocal laser endomicroscopy (pCLE), a confocal laser 
microscope is associated with a probe of multiple optical fibers allowing introduction (cross 
sectional diameter 1.2 mm) through the working channel of endoscopes. Confocal microscope 
can be located at the tip of the probe or in an external unit. This technique can show in vivo 
imaging at a cellular level with a video frame of 12 images per second, a lateral resolution of 3 
µm, an optical area of 1.13 mm² and a depth of focus of 0-50 µm.With this technology, clinicians 
can obtain in vivo and live imaging at the cellular level called “optical biopsies”. Furthermore, 
pCLE allows to see some details not visible during classic histology (due to fixation process) as 
blood flow (3), microorganisms (4) ... 
Probe based confocal laser endomicroscopy was fisrt used in gastroenterology (endobrachy 
oesophage, bilio-pancreatic lesions, colorectal polyps, inflammatory bowel disease…) (5). In 
pulmonary medicine pCLE came later to the light and is still an experimental technique but 
several studies are on going. Domains of interest are numerous: 
- 
Pulmonary nodules evaluation; 
- 
Interstitial lung dieases; 
- 
Lung and bronchus infections; 
- 
Lung rejection after transplantation (6); 
- 
Lung emphysema (7); 
- 
Mediastinal adenopathy (8); 
- 
Pleural malignancies ; 
Pleural effusion is a common finding in medical practice and often requires thoracocentesis and 
pleural biopsies to determine the underlying disease. However, thoracocentesis with pleural fluid 
cytology has limited sensitivity (up to 60% for repeated thoracocentesis) for a malignant pleural 
effusion diagnosis, one of the most common cause of pleural effusion (9–11). Therefore, 
thoracoscopy is often required, allowing direct pleural examination and biopsies with a sensitivity 
and a specificity of 94% and 100 % for malignant diseases(12,13). Nonetheless, this procedure is 
more invasive than a simple thoracocentesis. Recently, four studies demonstrated that pleural 
pCLE could be of value for the diagnosis of malignant pleural effusion. In 2018 Zirlik et al. 
showed that pCLE with fluoresceine performed in the pleural fluid after thoracocentesis was able 
to identify a malignant pleural effusion with 87 % sensitivity and 99% specificity compared 

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pleural fluid cytology (14). In 2019 Wijmans et al. showed that pCLE was able to guide pleural 
biopsies when the probe was passed through the biopsy needle during a thoracoscopy, an open 
thoracotomy or an ultra-sound or CT guided pleural biopsy when a malignant pleural 
mesothelioma was suspected(15). In 2021 our team published a prospective trial showing that 
pCLE during medial thoracoscopy was able to discriminate malignant from benign pleura(16,17). 
The same year, Sawada et al. showed that pCLE performed during surgery for lung cancer was 
able to identify a visceral pleural involvement with 100% sensitivity and 83% specificity(18). 
Considering these results and the low sensitivity of thoracocentesis for malignant pleural effusion 
identification, we conducted this proof of concept study to determine if mini-invasive close-
blinded pleural pCLE performed through a Boutin’s needle was feasible, safe and had a better 
sensitivity than thoracocentesis. 
 
 
Study design 
 
Exploratory study on a diagnostic procedure based on a cross-sectional study. We will 
prospectively include all patients referred to medical pleuroscopy for pleural effusion treatment 
or work-up. 
 
The intervention was divided into four parts realized by the two same experienced investigators: 
First, the thoracocentesis for pleural fluid sampling and pathological examination; Second, the 
mini-invasive pleural pCLE (Alveoflex ®, lateral resolution 3 µm, optical area 1.13 mm², depth 
of focus 0 - 50 µm) (Cellvizio®, Maunakea technologies®, Paris, France); Third, the invasive 
pleural pCLE through the thoracoscope; Fourth, the traditional medical thoracoscopy with pleural 
examinations, biopsies and talc poudrage if required. Two different thoracoscopes were used 
during this study: A Wolf thoracoscope with an outer diameter of 7 mm (Richard Wolf GmbH, 
Knittlingen Germany ®) and a Storz single puncture thoracoscope with an outer diameter of 10 
mm (Karl Storz GmbH, Tuttlingen, Germany ®).  For pleural staining, 5ml of fluorescein 10% 
were intravenously administrated 5 min before pCLE acquisition. The investigators were aware 
of the clinical history of the patients during the intervention as it happens in real clinical practice.  
 
For the mini-invasive pleural pCLE, the probe was blindly introduced in the pleural space 
through the Boutin’s needle (traditionally used in medical thoracoscopy to induce a 
pneumothorax before insertion of the trocar) until satisfying images were recorded.  At the same 
time, the two endoscopists rated 7 criteria (see the table 1 for the criteria and their interpretation). 
The part three (invasive pleural pCLE) started by the insertion of the trocar allowing to perform 
the medical thoracoscopy. For this part, the probe was gently applied on the parietal pleura 
through the working channel of the thoracoscope under vision control. If macroscopic 
abnormalities were noticed, pCLE was performed on the affected zones. In the absence of 
macroscopic abnormality, three random sites were selected. The same criteria were rated by the 
same two investigators. Finally, biopsies were systematically performed on the same sites for 
pathological diagnosis. 

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The assessed visual criteria were chosen in accordance with our previous publications and 
experience(16,17). 
 
 
 
 
Methods 
 
Inclusion/exclusion criteria 
Every patient referred to medical thoracoscopy for pleural effusion willing to participate and 
signing informed consent. 
Exclusion: unwilling to participate or unable to give informed consent; <18 years; pregnant 
woman; known allergy to fluorescein 
 
Controls: 
Pleural fluid cytological analysis 
 
Aims: 
To determine the feasibility and safety of mini invasive pleural pCLE assessment 
To determine the sensitivity of mini invasive pleural pCLE compared to pleural fluid cytological 
analysis 
 
Standard demographic, clinical, biological and endoscopic assessment 
Gender, age  
Medical background 
Smoking habit 
Current therapy 
Blood: CRP, Albumin, total proteins, Hemoglobin, Platelet count, White blood cells, LDH 
Pleural fluid: Cytological and bacteriological studies, LDH, Albumin, proteins 
 
 
Sample size 
No formal sample size calculation can be performed for such exploratory study. Every major 
patient referred for medical pleuroscopy will be proposed the study and screened if they give 
their informed consent. 
 
Statistical analysis 
The Fisher’s exact test was used to analyze the link between pCLE qualitative variables and the 
final histological diagnosis. No adjustments are made for multiple variable testing. 
 
 
 
 
 
 
 

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