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      Comparison of Real-Time Fluorescence Confocal Digital Microscopy With Hematoxylin-Eosin–Stained Sections of Core-Needle Biopsy Specimens

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          This diagnostic study investigates real-time acquisition of fluorescence confocal microscopy of interventional radiology–guided specimens in the radiology suite and compares accuracy of diagnoses with hematoxylin-eosin (H&E)–stained sections among patients undergoing core-needle biopsy.

          Key Points


          Can fluorescence confocal microscopy be used for immediate evaluation of interventional radiology–guided core-needle biopsy in real time at the bedside in the radiology suite?


          In this diagnostic study of 105 patients undergoing core-needle biopsy, fluorescence confocal microscopy images were acquired in a mean of 7 minutes, were similar to hematoxylin-eosin–stained tissue sections, and were of acceptable quality in 101 (96.2%). The accuracy of fluorescence confocal microscopy diagnosis compared with the criterion standard of hematoxylin-eosin–stained tissue sections was 95.2% for pathologist 1, 85.7% for pathologist 2, and 96.2% for consensus between them.


          These findings suggest that fluorescence confocal microscopy can be used for real-time bedside evaluation of interventional radiology–guided core-needle biopsy with high accuracy in a range of medical settings for procuring high-quality specimens in 1 hospital visit.



          Strategies to procure high-quality core-needle biopsy (CNB) specimens are critical for making basic tissue diagnoses and for ancillary testing.


          To investigate acquisition of fluorescence confocal microscopy (FCM) images of interventional radiology (IR)–guided CNB in real time in the radiology suite and to compare the accuracy of FCM diagnoses with those of hematoxylin-eosin (H&E)–stained CNB sections.

          Design, Setting, and Participants

          In this diagnostic study, FCM imaging of IR-guided CNBs was performed in the radiology suite at a major cancer center for patients with an imaging abnormality from August 1, 2016, to April 30, 2019. The time taken to acquire FCM images and the quality of FCM images based on percentage of interpretable tissue with optimal resolution was recorded. The FCM images were read by 2 pathologists and categorized as nondiagnostic, benign/atypical, or suspicious/malignant; these diagnoses were compared with those made using H&E-stained tissue sections. Cases with discrepant diagnosis were reassessed by the pathologists together for a consensus diagnosis. Data were analyzed from June 3 to July 19, 2019.


          Each IR-guided CNB was stained with 0.6mM acridine orange, subjected to FCM imaging, and then processed to generate H&E-stained sections.

          Main Outcomes and Measures

          Mean time taken for acquisition of FCM images, quality of FCM images based on interpretable percentage of the image, and accuracy of diagnostic categorization based on FCM images compared with H&E-stained sections.


          A total of 105 patients (57 male [54.3%]; mean [SD] age, 63 [13] years) underwent IR-guided CNBs in a mean (SD) of 7 (2) minutes each. The FCM images showed at least 20% of the tissue with optimal quality in 101 CNB specimens (96.2%). The FCM images were accurately interpreted by the 2 pathologists in 100 of 105 cases (95.2%) (2 false-positive and 3 false-negative) and 90 of 105 cases (85.7%) (6 false-positive and 9 false-negative). A reassessment of 14 discordant diagnoses resulted in consensus diagnoses that were accurate in 101 of 105 cases (96.2%) (1 false-positive and 3 false-negative).

          Conclusions and Relevance

          The ease of acquisition of FCM images of acceptable quality and the high accuracy of the diagnoses suggest that FCM may be useful for rapid evaluation of IR-guided CNBs. This approach warrants further investigation.

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          Most cited references 33

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          Quality improvement guidelines for percutaneous needle biopsy.

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            Assessment of breast pathologies using nonlinear microscopy.

            Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was κ = 0.88 for NLM and κ = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.
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              Microscopy with ultraviolet surface excitation for rapid slide-free histology

              Histologic examination of tissues is central to the diagnosis and management of neoplasms and many other diseases, and is a foundational technique for preclinical and basic research. However, commonly used bright-field microscopy requires prior preparation of micrometre-thick tissue sections mounted on glass slides, a process that can require hours or days, that contributes to cost, and that delays access to critical information. Here, we introduce a simple, non-destructive slide-free technique that within minutes provides high-resolution diagnostic histological images resembling those obtained from conventional haematoxylin-and-eosin-histology. The approach, which we named microscopy with ultraviolet surface excitation (MUSE), can also generate shape and colour-contrast information. MUSE relies on ~280-nm ultraviolet light to restrict the excitation of conventional fluorescent stains to tissue surfaces, and it has no significant effects on downstream molecular assays (including fluorescence in situ hybridization and RNA-seq). MUSE promises to improve the speed and efficiency of patient care in both state-of-the-art and low-resource settings, and to provide opportunities for rapid histology in research.

                Author and article information

                JAMA Netw Open
                JAMA Netw Open
                JAMA Netw Open
                JAMA Network Open
                American Medical Association
                5 March 2020
                March 2020
                5 March 2020
                : 3
                : 3
                [1 ]Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston
                [2 ]Department of Radiology, Scripps Mercy Hospital, San Diego, California
                [3 ]Department of Interventional Radiology, The University of Texas MD Anderson Cancer Center, Houston
                [4 ]Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston
                [5 ]Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, Houston
                [6 ]Oregon Health and Science University Knight Cancer Institute, Portland
                [7 ]Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston
                Author notes
                Article Information
                Accepted for Publication:
                Open Access: CC-BY License JAMA Network Open
                Corresponding Author: Savitri Krishnamurthy, MD, Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030 ( skrishna@ ).
                Author Contributions:
                Concept and design:
                Acquisition, analysis, or interpretation of data:
                Drafting of the manuscript:
                Critical revision of the manuscript for important intellectual content:
                Statistical analysis:
                Obtained funding:
                Administrative, technical, or material support:
                Conflict of Interest Disclosures:
                Role of the Funder/Sponsor:
                Additional Contributions:
                Copyright 2020 Krishnamurthy S et al. JAMA Network Open.

                This is an open access article distributed under the terms of the CC-BY License.

                Original Investigation
                Online Only
                Pathology and Laboratory Medicine


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