Probe Based Confocal Laser Endomicroscopy During Thoracoscopy for Pleural Malignancies Diagnosis.
Conditions: Pleural Carcinomatosis
Sponsor: University of Liege

Probe based confocal laser endomicroscopy for pleural malignancies diagnosis. 
clinicaltrial.org NCT03805971 
12/02/2019

Study protocol 
This is an ethically approved (clinicaltrial.org NCT03805971) single center cross-sectional study. 
Every patient ≥ 18 years referred for a medical thoracoscopy (pleural effusion work up, recurrent 
pneumothorax, talc pleurodesis….) was selected for the present study between 06/2018 and 
09/2019. Exclusion criteria were pregnancy  and known allergy to fluorescein. The thoracoscopy was 
performed under sedation (allowing spontaneaous breathing) by two experienced investigators with 
a systematic procedure. The patient was positioned in lateral decubitus during the intervention. After 
chest ultrasound examination, a pneumothorax was induced with the Boutin trocar and one entry 
port was performed  though the chest wall. Two different thoracoscopes were used during this 
study: 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 ®). Five milliliters of fluorescein (10%) were intravenously administrated 5 
minutes before image acquisition. After that, the pleural cavity was examined macroscopically. Then 
the pCLE probe (Alveoflex ®, lateral resolution 3 µm, optical area 1.13 mm², depth of focus 0 - 50 µm) 
(Cellvizio®, Maunakea technologies®, Paris, France) was gently placed on the parietal pleura and 
videos were recorded.  If macroscopic abnormalities were noticed, pCLE was performed on affected 
zones, if not, three random sites were selected. Finally, biopsies were systematically performed on 
the same sites for comparison with optical biopsies. During the thoracoscopy, the investigators rated 
the quality of the pCLE acquisitions (good, acceptable, low).  
After the image acquisition, another investigator (not included in those who rated the quality of the 
acquisition) selected the five best representative images for further analysis and comparison to 
histological samples. According to our previous experience, 11 criteria were selected to assess the 
ability of pCLE to distinguish benign from malignant pleura. The abnormal tissular architecture was 
defined by the identification of tissular/cellular structures not known to belong to the normal pleura  
(cell clusters, dark clumps, glands, cell cordons, dysmorphic cells, papillar architecture…).  The 
absence of abnormal architecture was defined by the identification of normal pleural cellular and 
tissular elements as previously reported: the mesothelial monolayer with homogenous and regularly 
distributed cells; the fibro-adipose connective tissue and its regular and homogeneous 
vascularization; muscular fibers. The cells homogeneity in size, fluorescence and shape was 
subjectively assessed by the investigator. The mean cellular sized was assessed on a full field of view 
(1.13 mm²). The presence of dysplastic vessels was assessed, as described by Cannizzaro et al., by the 
identification of vascular leaks (fluorescein leakage outside the vessels), tortuous vessels or giant 
vessel. For the maximal vascular diameter, we selected the largest vessels among the available pCLE 
images. For the vascular density, we measured the max number of vessels/image (full field of view) 
for every patient on the available images. For our study, we selected only the vessels with a length of 
at least 50 µm.  The connective tissue fibers were classified as anarchic or organized. Anarchic 
connective tissue fibers were defined as irregular in shape or direction, coarse, without a well-
defined architecture. Organized connective tissue fibers were defined as regular in shape and 
direction with a well-defined architecture. The chia seed sign defines the presence on a full field of 
view of the mesothelial monolayer with well defined, homogeneous and organized mesothelial cells. 
The criteria were not scored if assessable elements for their interpretation were missing. Every 
patient included gave informed consent. 
Statistical analysis plan 
The Fisher’s exact test was used to analyze the link between pCLE qualitative variables and the final 
histological diagnosis (p < 0.05 for statistical significance). For quantitative variables, the unpaired T-
test (2 sided p-value, p < 0.05) was used.