Study population
The study was conducted at Beijing Tsinghua Changgung Hospital between April and December 2021. Patients aged > 18 years and were indicated for hysterectomy were consecutively enrolled in this study. All the patients signed informed consent before surgery. The study was approved by the Ethics Committee of Beijing Tsinghua Changgung Hospital(No.22221-4-01).
Image acquisition and dye application method
After hysterectomy, the isolated specimen was immediately transferred to the observation table. Then it specimen was dissected from the anterior wall of the uterus (Fig. 1), and 0.1% sodium fluorescein [sodium fluorescein injection: 5 ml:0.5 g (10%; Lishede, Beijing)] was evenly sprayed on the endometrium, myometrium, endocervical canal, and surface of the external os of the cervix. After 5–10 min, we observed the resected specimen using pCLE (Fig. 2), and the flexible confocal miniprobe was placed directly perpendicular to the tissue. Real-time video recording and diagnosis were performed on the displayed images. Video recording (6 frames per second) of the image acquisition process was performed to facilitate repeat viewing and to correct image recognition while the typical images were saved.
The primary objective of this study was to observe the endometrium and analyze the microscopic morphological characteristics of EC, atypical endometrial hyperplasia, endometrial hyperplasia without atypia, and normal endometrium, and compare with histopathological characteristics. The secondary objective was to observe the myometrium, endocervical canal, and surface of the external os of the cervix.
All the videos and images were saved, and the images were analyzed and compared with the histopathological images.
Equipment and image acquisition
Images were acquired using a CLE-1000 equipment (laser scanning unit LSU-1000, confocal probe U-300, magnification). The probe had an insertion part with a maximum outer diameter of 2.6 mm and length of 3 m, and could be inserted to a depth of 40 μm below the tissue surface for observation. The LSU generated a laser beam with a wavelength of 488 nm that, after a series of transmissions and deflections, was focused on the proximal end of the probe and transmitted to the tissue sample through the optic fiber and microscope at the distal end. The microscope collected the fluorescence scattered by the tissue, which was then passed through the optical fiber bundle and a series of beam coupling and transmission devices. It was received by the photoelectric conversion device of the LSU and converted into current signals. The signals were processed by specific algorithms and software to generate real-time images of the tissue, which were displayed on the computer screen. The imaging software could perform single-frame capture and video recording of the acquired images for later analysis.
Histopathological analysis
Two gynecologists, who were blinded to the patient’s condition or previous pathological diagnosis via curettage, observed the pCLE videos for all specimens, recorded the diagnoses of the images, and moved the lesions from the observation area to make sections. The pathological diagnosis of the endometrium was regarded as the gold standard. Mainly referring to the 2014 World Health Organization classification of tumors of female reproductive organs, abnormal endometrium is divided into (1) endometrial hyperplasia without atypia (EH), (2) atypical hyperplasia (AH), and (3) EC, according to the presence or absence of architectural and cellular atypia. The diagnostic results of the images were compared with the pathological results of the endometrial tissues. Benign endometrium and EH were regarded as negative, and AH and EC as positive. The diagnostic concordance rate of pCLE was evaluated. (Fig. 3)
Statistical analysis
Statistical analyses were performed using SPSS 13.0 (Chicago, IL, USA). The statistical methods used in our study aimed to evaluate the diagnostic accuracy of the tests in comparison with the gold standard method. The sensitivity, specificity, positive predictive value, and negative predictive value were determined. Histopathological diagnostic findings were considered the gold standard for assessing the diagnostic accuracy of pCLE.
Diagnostic accuracy = Number of true-positive and true-negative cases diagnosed by pCLE / total number of cases × 100%.
Sensitivity = True positive cases diagnosed by pCLE / positive cases diagnosed by pathology × 100%.
Specificity = True negative cases diagnosed by pCLE / negative cases diagnosed by pathology × 100%.
Positive predictive value = True positive cases diagnosed by pCLE / positive cases diagnosed by pCLE × 100%.
Negative predictive value = True negative cases diagnosed by pCLE / negative cases diagnosed by pCLE × 100%.