RESUMO
Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 µm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.
Assuntos
Carcinoma de Mama in situ/patologia , Neoplasias da Mama/patologia , Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/patologia , Margens de Excisão , Laranja de Acridina/química , Mama/citologia , Mama/imunologia , Mama/cirurgia , Carcinoma de Mama in situ/diagnóstico , Carcinoma de Mama in situ/imunologia , Carcinoma de Mama in situ/cirurgia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/imunologia , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/imunologia , Carcinoma Ductal de Mama/cirurgia , Carcinoma Lobular/diagnóstico , Carcinoma Lobular/imunologia , Carcinoma Lobular/cirurgia , Corantes/química , Feminino , Corantes Fluorescentes/química , Humanos , Imageamento Tridimensional , Período Intraoperatório , Mastectomia , Mastectomia Segmentar , Microscopia de Fluorescência , Invasividade Neoplásica , Microscopia Óptica não Linear , Tratamentos com Preservação do Órgão , Rodaminas/químicaAssuntos
Oxalato de Cálcio/análise , Derivação Gástrica/efeitos adversos , Hiperoxalúria/etiologia , Imageamento Tridimensional/métodos , Rim/química , Insuficiência Renal/etiologia , Idoso , Biópsia , Cristalização , Feminino , Humanos , Hiperoxalúria/metabolismo , Hiperoxalúria/patologia , Rim/patologia , Valor Preditivo dos Testes , Insuficiência Renal/metabolismo , Insuficiência Renal/patologiaRESUMO
Histology is the study of the structure of biological tissue using microscopy techniques. As digital imaging technology advances, high resolution microscopy of large tissue volumes is becoming feasible; however, new interactive tools are needed to explore and analyze the enormous datasets. In this paper we present a visualization framework that specifically targets interactive examination of arbitrarily large image stacks. Our framework is built upon two core techniques: display-aware processing and GPU-accelerated texture compression. With display-aware processing, only the currently visible image tiles are fetched and aligned on-the-fly, reducing memory bandwidth and minimizing the need for time-consuming global pre-processing. Our novel texture compression scheme for GPUs is tailored for quick browsing of image stacks. We evaluate the usability of our viewer for two histology applications: digital pathology and visualization of neural structure at nanoscale-resolution in serial electron micrographs.
Assuntos
Gráficos por Computador , Técnicas Histológicas/estatística & dados numéricos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Humanos , Microscopia Eletrônica de Transmissão/estatística & dados numéricosRESUMO
Mohs surgery uses en face frozen section analysis (FSA) with complete margin examination for the excision of select basal cell carcinomas (BCC), obtaining excellent cosmetic outcomes and extremely low recurrence rates. However, Mohs with FSA is time-consuming because of the need to iteratively perform cryosectioning on sequential excisions. Fluorescent microscopies can image tissue specimens without requiring physical sectioning, potentially reducing the time to perform Mohs surgery. We demonstrate a protocol for nonlinear microscopy (NLM) imaging of surgical specimens that combines dual agent staining, virtual H&E rendering, and video rate imaging. We also introduce a novel protocol that enables micron-level co-registration of NLM images with FSA histology, and demonstrate that NLM can reproduce similar features similar to FSA in BCC specimens with both negative and positive surgical margins. We show that the fluorescent labels can be extracted with conventional vacuum infiltration processing, enabling subsequent immunohistochemistry on fluorescently labeled tissue. This protocol can also be applied to evaluate the performance of NLM compared with FSA in a wide range of pathologies for intraoperative consultation.
RESUMO
IMPORTANCE: Extramammary Paget disease (EMPD), a rare intraepithelial adenocarcinoma, poses a therapeutic challenge with high postoperative recurrence rates and a limited number of effective local treatment options. OBJECTIVE: To describe the use and efficacy of a topical combination of fluorouracil and calcipotriene as a palliative therapy for refractory EMPD. DESIGN, SETTING, AND PARTICIPANTS: This retrospective case series of 3 women with recurrent, refractory EMPD was conducted at Beth Israel Deaconess Medical Center, Boston, Massachusetts and Washington University School of Medicine, St Louis, Missouri. All patients were treated with a 1:1 mixture of fluorouracil, 5%, cream and calcipotriene, 0.005%, cream or ointment. MAIN OUTCOMES AND MEASURES: Clinical and histopathological findings. RESULTS: All 3 women (1 in her 50s, 2 in their 70s) presented with recurrent EMPD (vulvar, perianal, and perioral) after surgery and/or irradiation, and their EMPD was refractory to treatment with imiquimod, 5%, cream. Owing to disease progression and/or intolerable adverse effects from imiquimod, the patients began treatment with a 1:1 mixture of fluorouracil, 5%, cream and calcipotriene, 0.005%, cream. This treatment, which was well tolerated, was followed by clinical improvement in symptoms and appearance of the lesions in all 3 cases and histopathological signs of decreased tumor burden in 2 cases. Patients applied the combination topical therapy to affected areas with differing frequencies, ranging from 1 to 2 days per month to 4 consecutive days every 2 weeks. CONCLUSIONS AND RELEVANCE: Extramammary Paget disease frequently recurs even after aggressive surgical management and can be refractory to many topical and locoregional therapies. Palliative treatment with a combination of fluorouracil and calcipotriene may be a viable option for patients with recurrent, refractory EMPD.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Doença de Paget Extramamária/tratamento farmacológico , Cuidados Paliativos/métodos , Neoplasias Cutâneas/tratamento farmacológico , Administração Tópica , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Calcitriol/administração & dosagem , Calcitriol/análogos & derivados , Feminino , Fluoruracila/administração & dosagem , Humanos , Imiquimode/administração & dosagem , Pessoa de Meia-Idade , Doença de Paget Extramamária/patologia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Scleroderma vasculopathy and ANCA (antineutrophil cytoplasmic antibodies)-associated glomerulonephritis have rarely been reported to occur simultaneously in one patient. Herein, we report a patient who presented with a classic constellation of clinical and laboratory findings of systemic scleroderma and was subsequently found to be positive for p-ANCA. Two renal biopsies, performed 5 months apart, demonstrated typical changes of the two entities in both acute and "healed" phases, which were analyzed by computer mapping techniques. The two renal biopsies were serially sectioned and stained routinely, and with CD31 and CD34 as endothelial markers. The slides were digitized, aligned and analyzed. Each glomerular tuft was sequentially studied in terms of total area (µm2) and each biopsy was individually profiled. All arterial vessels were sequentially studied with whole vessel and luminal areas delineated and ratios calculated. The initial biopsy contained 32 glomeruli almost all with extensive fibrinoid necrosis and destruction of the capillary network. The arterial vessels (interlobular and arcuate) showed intimal edema with luminal occlusion. CD31/CD34 stains showed variable endothelial intactness but demonstrated the luminal size shifts. The second biopsy had 37 glomeruli that were either segmentally or globally sclerotic with no active changes. The vessels were now normally patent. Each glomerular tuft and arterial vessel in both biopsies was analyzed as a serial section histogram documenting these changes. These studies depict the rare occurrence of two entities together, the scleroderma kidney vasculopathy and the glomerulonephritis of ANCA-associated vasculitis syndrome both in an acute and healing phase, profiled by computer mapping techniques.
RESUMO
Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment. Microscopy with ultraviolet surface excitation (MUSE) is a low-cost optical sectioning technique using ultraviolet illumination which limits fluorescence excitation to the specimen surface. In this paper, we characterize MUSE using high incident angle, water immersion illumination to improve sectioning. Propidium iodide is used as a nuclear stain and eosin yellow as a counterstain. Histologic features of specimens using MUSE, nonlinear microscopy (NLM) and conventional hematoxylin and eosin (H&E) histology were evaluated by pathologists to assess potential application in Mohs surgery for skin cancer and lumpectomy for breast cancer. MUSE images of basal cell carcinoma showed high correspondence with frozen section H&E histology, suggesting that MUSE may be applicable to Mohs surgery. However, correspondence in breast tissue between MUSE and paraffin embedded H&E histology was limited due to the thicker optical sectioning in MUSE, suggesting that further development is needed for breast surgical applications. We further demonstrate that the transverse image resolution of MUSE is limited by the optical sectioning thickness and use co-registered NLM to quantify the improvement in MUSE optical sectioning from high incident angle water immersion illumination.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Microscopia Ultravioleta/métodos , Neoplasias Cutâneas/diagnóstico por imagem , Neoplasias Cutâneas/patologia , Neoplasias da Mama/cirurgia , Desenho de Equipamento , Feminino , Técnicas Histológicas , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Microscopia Ultravioleta/instrumentação , Neoplasias Cutâneas/cirurgiaRESUMO
The ability to histologically assess surgical specimens in real-time is a long-standing challenge in cancer surgery, including applications such as breast conserving therapy (BCT). Up to 40% of women treated with BCT for breast cancer require a repeat surgery due to postoperative histological findings of close or positive surgical margins using conventional formalin fixed paraffin embedded histology. Imaging technologies such as nonlinear microscopy (NLM), combined with exogenous fluorophores can rapidly provide virtual H&E imaging of surgical specimens without requiring microtome sectioning, facilitating intraoperative assessment of margin status. However, the large volume of typical surgical excisions combined with the need for rapid assessment, make comprehensive cellular resolution margin assessment during surgery challenging. To address this limitation, we developed a multiscale, real-time microscope with variable magnification NLM and real-time, co-registered position display using a widefield white light imaging system. Margin assessment can be performed rapidly under operator guidance to image specific regions of interest located using widefield imaging. Using simulated surgical margins dissected from human breast excisions, we demonstrate that multi-centimeter margins can be comprehensively imaged at cellular resolution, enabling intraoperative margin assessment. These methods are consistent with pathology assessment performed using frozen section analysis (FSA), however NLM enables faster and more comprehensive assessment of surgical specimens because imaging can be performed without freezing and cryo-sectioning. Therefore, NLM methods have the potential to be applied to a wide range of intra-operative applications.
RESUMO
Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Interpretação de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Mama/diagnóstico por imagem , Desenho de Equipamento , Feminino , Humanos , Microscopia Confocal/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentaçãoRESUMO
We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Mama/diagnóstico por imagem , Amarelo de Eosina-(YS)/química , Hematoxilina/química , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Transiluminação/métodos , Algoritmos , Mama/patologia , Neoplasias da Mama/patologia , Feminino , HumanosRESUMO
OBJECTIVE: To assess the effectiveness of fine needle aspiration biopsy (FNAB), with and without immunohistochemistry (IHC), in the management of solid intraocular tumors. STUDY DESIGN: Thirty-three consecutive adults undergoing FNAB of suspected intraocular tumors were studied. Clinical, cytologic and histologic diagnoses were correlated. The positive predictive value, sensitivity and specificity of FNAB for detecting malignancy, the effect of lHC on the final cytologic diagnosis and the number of patients in whom clinical management was altered as a result of cytologic evaluation were determined. RESULTS: The positive predictive value was 96% with and 93% without adjunct IHC. The sensitivity and specificity of FNAB for detecting malignancy were 96% and 83%, respectively, with IHC. Without IHC, the sensitivity was unaltered, but the specificity was 67%. IHC confirmed the morphologic diagnosis in 75% of cases, made a diagnosis in 12.5% and changed a malignant diagnosis from carcinoma to melanoma in 6% of cases. The planned management was changed by the FNAB findings in 24% of patients. In 3 patients (9%), IHC was essential for diagnosis and management. No patients exhibited local tumor dissemination or recurrence associated with the biopsy. CONCLUSION: FNAB is a safe, sensitive and specific method of establishing a tissue diagnosis in a subset of patients with solid intraocular tumors. The routine use of immunohistochemical stain ing increases the diagnostic utility of the technique and may change clinical management.