Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Summary

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

Abstract

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory¹ and gastrointestinal tracts,^2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.^7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.⁷

The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.¹¹ Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent—most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.¹² Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.¹⁰ Recent availability of a < 1 mm imaging probe¹³ opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities¹⁴ that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides¹⁵ and antibodies for endoscopic imaging of disease processes with molecular specificity.

Protocol

1. Patient Preparation

1. Consent patient scheduled for diagnostic cystoscopy and other endourological procedures such as transurethral resection of bladder tumor (TURBT) for CLE. Include in the consent a description of the use of intravesical and/or intravenous fluorescein as the contrast agent. Inquire history of hypersensitivity reaction to fluorescein.
2. Patient is positioned for cystoscopy (typically in lithotomy position) and prepared in a sterile fashion.
3. Proceed with standard white light cystoscopy (WLC) with a rigid or flexible cystoscope through the urethra.
4. Survey all regions of the bladder under white light (Figure 1) and note regions of interest for CLE investigation.
5. In addition to WLC, the bladder may be imaged with other macroscopic optical imaging modalities including fluorescence cystoscopy (Figure 2A) or narrow band imaging (Figure 2B) to identify additional areas of suspicion followed by characterization with CLE.
6. Use a cautery electrode or resection loop to place a small cautery mark adjacent to the regions of interest that will both be imaged and biopsied for pathological confirmation. The cautery mark facilitates re-localization for subsequent CLE imaging and biopsy.

2. Contrast Agent

1. Intravesical instillation of contrast agent
   1. Prepare by diluting clinical grade 10% fluorescein sodium in sterile normal saline (0.9% NaCl) to desired concentration. For example, prepare 400 ml of 0.1% fluorescein by diluting 4 ml of 10% fluorescein in 396 ml of normal saline.
   2. Insert a sterile urinary catheter into the bladder and instill diluted contrast agent by gravity using a 60 ml catheter-tip syringe.
   3. Clamp the urinary catheter to hold contrast agent indwelling for 5 min.
   4. Drain contrast agent from bladder. Use the 60 ml catheter-tip syringe to wash out residual contrast from the bladder using normal saline, about 240-300 ml.
   5. Proceed to CLE imaging.
2. Intravenous injection of fluorescein
   1. Draw 1.0 ml of fluorescein into syringe. Attach syringe to intravenous access and inject 0.5 - 1.0 ml of fluorescein intravenously as a bolus. Flush line with normal saline. This is typically done by the anesthesia team in the operating room.
   2. Proceed to CLE imaging.

3. CLE Imaging

1. Remove the sterilized CLE imaging probe (Cellvizio) from its packaging and connect it to the laser scanning unit (Mauna Kea Technologies, Paris, France)
2. Warm up and calibrate the imaging probe by following the manufacturer's instructions.
3. Re-insert the cystoscope or resectoscope into the bladder. Use a 0 degree lens with the rigid cystoscope.
4. Advance the CLE imaging probe along the working channel of the cystoscope with the tip just beyond the cystoscope.
5. Under white light, locate the previously marked regions of interest. Use additional normal saline irrigation as needed for visualization of the mucosa and maintain moderate distension of the bladder.
6. Manipulate CLE imaging probe with the operator's hand for direct en face contact of the regions of interest. For optical sectioning of the regions of interest, gently increase and decrease pressure to the imaging probe while maintaining direct contact.
   1. To reach tumors located at the anterior bladder, a standard Albarran bridge may help facilitate deflection of the imaging probe for direct contact with the region of interest (Figure 2C).
7. Common regions of interest include papillary tumor, non-papillary tumor, transition between normal-appearing mucosa and tumor, and erythematous patches, which can all be imaged using either intravesical or intravenous fluorescein administration. Intravenous fluorescein is required for imaging of the resection bed, prostatic urethra, and penile urethra.
8. As the imaging probe scans region of interest, record images for future analysis. Note overall cellular morphology, boundary, organization, and presence of neo-angiogenesis.
9. Upon completion of imaging, remove CLE imaging probe from working channel.
10. If desired, obtain cold-cup biopsies from corresponding regions of interest that were scanned by CLE. Use cautery markings as landmarks for co-registration of the imaged areas that are biopsied.
11. Complete TURBT per routine. In patients who received intravenous fluorescein, consider using continuous flow resectoscopes to improve visualization during resection, as fluorescein is excreted by the kidney into the bladder approximately 5 min after intravenous administration.
12. Retrieve saved image recordings from confocal processor and conduct image analysis.

Results

CLE images are saved as grayscale video sequence files at 12 frames per second. Image interpretation is done in real time and may be used to impact clinical decision making under an investigational protocol. Offline analysis, which includes reviewing of the video sequence, additional image processing such as mosaicing,⁷ and comparison with standard pathology, are important during the learning curve phase associated with the technology. Figures 1B and 1C are representative CLE images obtained from two different...

Discussion

Attaining and maintaining solid en face contact between the imaging probe and the bladder mucosa is the most critical step in acquiring optimal image quality. There is approximately a 3-5 patient learning curve to develop the dexterity to manipulate the imaging probe and to hold the probe steady during image acquisition. Additionally, as this procedure is conducted in vivo, patient movements (i.e. respiratory) and vascular pulsations may affect the imaging probe contact with the bladder....

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number	Comments (optional)
Cellvizio 100 Series	Mauna Kea Technologies	100 Series	Includes confocal processor and LSU: F400-v2 at 488 nm
Cellvizio Confocal Miniprobe	Mauna Kea Technologies	Gastroflex UHD
AK-FLUOR 10%	Akorn, Inc.	NDC 17478-253-10