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Recently a confocal laser microscopy scanner became available for evaluation. The technique uses a confocal microscope and allows observation of the tissue in a spectrum between gross morphology and a sub-cellular level that approaches the resolution of a regular 5x objective. Histological images of the superficial layers of fresh thick tissue can be generated for a scanning area of 2.

HS was recently tested in a clinical study on skin cancer specimens with encouraging results [ 15 ].

Similarly to the intraoperative situation, quality and reliability of diagnosis with image guided biopsies relies on the quality of the technique, and experience of the performing clinician and the pathologist. The aim of our investigation was to assess image quality and correct diagnosis of human breast cancer tissue in core needle biopsies by using the HS, and to further evaluate the practicability and reliability of the device. For this purpose, two independent experienced pathologists read and interpreted the images obtained with the HS scanner and the images obtained after digitalisation of the routinely processed core biopsies.

The primary endpoint was to evaluate the correspondence of breast cancer diagnosis between assessment of confocal HS images and gold standard histological images. We examined 23 ultrasound guided core needle biopsies and one tomosynthesis guided vacuum biopsy, adding up to 24 cases of breast tumors which presented as suspicious for cancer in female patients. After examination of the patient by a consultant medical doctor and sonographic or mammographic detection of a breast lesion suspicious for breast cancer, a biopsy with two samples was taken to confirm the diagnosis histologically.

Taking two or more biopsy samples from a suspicious lesion is established in the clinical routine for sensitivity improvement. General informed consent was obtained from each patient before any diagnostic procedure was initiated. After taking biopsy samples from a suspicious lesion of the breast, the fresh biopsy specimen was immediately processed for HS imaging. The specimen was then ready for imaging procedure, which provided a preview and a maximum-resolution picture.

Techniques in Confocal Microscopy - 1st Edition

Staining was performed with Acridine Orange solution 0. The device integrates a computer with an image display monitor and relies on the imaging of laser scanning confocal fluorescence microscopy. Staining with a fluorescence dye was performed before on-site scanning. The encircled areas indicate invasive breast cancer location as detected by the pathologists in both images using the full resolution zooming feature to reveal morphological details.

Zoomed details of the microscopic scanner images with artificial coloring of the grey values: normal breast tissue left side and invasive carcinoma right side. Two pathologists from different centers independently evaluated the two HS acquire mode images of every specimen. The evaluation results from both HS images as well as those from the standard histology were allocated to one of the different categories of breast cancer detection correspondent to the B-Classification as an established reporting system for minimal invasive biopsies 0: No diagnosis possible; 1: Normal tissue; 2: Benign lesion; 3: Indeterminate; 4: Suspicious of malignancy; 5: Malignancy [ 16 ].

Broad sense correspondence; NO mismatch, ND could not be determined. Participants granted a written consent to participate.

An introduction to confocal microscopy with the OLYMPUS FV10i

Two pictures in the acquired HS mode were obtained for 18 specimens. In six samples, only one image was suitable for analysis. In two cases, poor quality of the images led to the exclusion. Limited performing time was the reason in four cases, e.

A Level Biology Laser Scanning Confocal Microscopy

The images were analyzed by two independent pathologists yielding 84 diagnoses in total Tables 1 and 2. In 80 cases, pathological diagnosis corresponded to the B-classification. In four cases, pathological diagnosis was not obtainable in the first image due to limited specimen quality.

After re-positioning the sparse tissue, the second image allowed an adequate diagnosis except in one case. Four mismatches lack of correspondence occurred, one of them likely due to limited quality of the specimen. The final gold-standard assessments resulted in 20 diagnoses of invasive cancer, two carcinoma-in-situ DCIS and two benign lesions. In the final histopathological diagnosis, 13 biopsy specimens were diagnosed as invasive carcinoma of no special type NST.

In four cases, lobular invasive cancer was diagnosed, in one specimen an invasive- apocrine carcinoma, one invasive- mucinous carcinoma, and one micropapillary carcinoma. DCIS and benign lesions were each detected in two specimens. We recognized four mismatches in different cancer subtypes: two in lobular invasive cancer, one in invasive- mucinous carcinoma, and one in carcinoma of NST.

The Light Microscope Can Resolve Details 0.2 μm Apart

HE stains and high magnification histological appearance of two cases. Pictures show the negligibly small effect of the preparation process for scanning on the final pathologic routine: On-site scanning procedure did not interfere with the subsequent control immunohistochemical staining process. Immunohistochemical stains of two cases. Case invasive ductal NST carcinoma. B HE stain, formalin fixation after confocal microscopy. Case high grade DCIS. This report shows a promising approach to achieve fast and on-site diagnosis of high quality images obtained from the surface of fresh CNB.


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The method does not interfere with the standard of care workflow and therefore also has great potential to support surgical margin assessment in breast conserving therapy. Using this innovative approach, histology grade images from the entire surface of the resection margins can be obtained without loss of tissue due to freezing and cutting artefacts.

Furthermore, the subsequent procedure of fixation, histologic and immunologic examination of the specimen is not affected.


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HS images eliminate the need for mosaicking of multiple images, and therefore misinterpretation caused by gaps or overlapping can be excluded. However, earlier reports also showed accurate and reliable evaluation of confocal microscopy images by surgeons [ 12 ]. Misinterpretation of the lobular cancer was seen in the analysis of pathologist A, while pathologist B had a narrow sense correspondence for these images. The other two mismatches were found in cases where only one image of the specimen was available to the pathologists. Employing the HS scanning technique, this study clearly demonstrates that quality monitoring of the biopsy tissue on-site is reliable, which can reduce the rate of false negative results or re-biopsies.

As usually both sides of the tissue sample are imaged, we also see an advantage in that approach compared to the routine histological examination. With a high level of correspondence between two independent pathologists, the present study demonstrates that carcinoma of the breast can be diagnosed with a high accuracy in biopsy specimen using HS images. The presented study has some limitations.

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We analyzed a limited number of cases, and included only highly suspicious lesions. Further prospective studies are underway to substantiate our results on a larger cohort and variety of breast lesions and furthermore, to evaluate performance of the device on surgical specimen. It is simple to use, cost- and time-efficient and has a great potential to be adopted for routine use in intra-operative margin assessment.

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We are grateful to all patients who participated in this research. We would also like to acknowledge the essential support of colleges from the research department. All authors made substantial contributions to the study, they have approved the current version and agreed publication. Support was provided for study assessment, histopathological analyses, and data administration. September 18, This 's Fruad Compnay.

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