Stefano Forti

Distinct Patterns of Calcium Transients During Early and Delayed Afterdepolarizations Induced by Isoproterenol in Ventricular Myocytes

BACKGROUND The relation between early afterdepolarizations (EADs) and changes in intracellular Ca2+ concentration ([Ca2+]i) is still unclear. In the present study, we compared spatiotemporal changes in [Ca2+]i related to EADs and delayed afterdepolarizations (DADs) induced by isoproterenol. METHODS AND RESULTS Isolated patch-clamped guinea pig ventricular myocytes, loaded with fluo-3 acetoxymethyl ester (fluo-3 AM), were paced at 0.1 to 2 Hz. Isoproterenol (100 nmol/L) caused alterations in both phase 2 and phase 4 of the action potential (AP), consistent with EADs and DADs, respectively. During EADs (n = 16), similar to driven APs, increases in [Ca2+]i occurred simultaneously throughout the cell, whereas during DADs (n = 25), they originated in discrete cell sites and propagated as a wave. This difference was confirmed by analysis of eight EADs and DADs coupled to the same beat. Ca2+ transient linked to EADs reached a peak relative fluorescence level (expressed as percentage of the maximal level reached during the last stimulated beat) that was always higher than that reached during the DADs (77 +/- 3% versus 64 +/- 2%, P < .001). Spatial heterogeneity of Ca2+ transients was assessed by the maximal time interval between peaks monitored in different cell regions; this time lag was always greater during DADs than during EADs (290 versus 40 milliseconds, P = .006). CONCLUSIONS The present study had two main findings. First, even very modest notches occurring during the plateau of the AP may be accompanied by a marked secondary increase in [Ca2+]i. Second, these Ca2+ transients occurring during EADs are synchronous throughout the cell and differ significantly from those observed under identical conditions during DADs.

Digital storage of glass slides for quality assurance in histopathology and cytopathology

Proficiency testing programmes for measuring screening skills in pathology are mainly conducted using conventional glass microscope slides. However, the availability of robotic microscopes allows an entire conventional slide to be digitized. Our experiments have shown that, using a widely available robotized microscope and a PC, the image of a single field may be acquired in 2 s on average, including stage movements, autofocus and storage. Digitizing an entire slide, a fully automated procedure, takes up to 8 h. If the image of each field is compressed at an appropriate quality level (a compression ratio of, say, 35:1) it requires about 40 kByte to be stored, resulting in a total storage requirement of about 600 MByte per slide. Thus one CD-ROM can be used to store one virtual slide, as well as a self-installing program to provide a microscope simulator facility. This allows pathologists to examine the virtual case from their computer in a similar manner to looking at a glass slide on a conventional microscope. This permits a new, computer-based approach to proficiency testing in histopathology and cytopathology. Use of virtual slides should encourage the diffusion of national quality assurance programmes, which at present suffer from certain organizational and logistical limitations.

Expert pathology consultation through the Internet: melanoma versus benign melanocytic tumours

Twenty consecutive cases of melanocytic lesions were chosen from the archives of the Institute of Anatomic Pathology at Santa Chiara Hospital, Trento. Representative images were acquired at a spatial resolution of 512×512 pixels, saved in JPEG format and delivered to the remote pathologist by multimedia Internet electronic mail. Six cases were diagnosed as benign melanocytic lesions by the local pathologist. Of the 20 cases transmitted, each with an average of 5.3 images, the remote pathologist suggested a diagnosis of malignancy in nine cases while 10 cases were thought to be benign. In one case the images were not considered sufficient for diagnosis. Overall, the diagnostic agreement between local and remote pathologist was 79%(κ=0.58, P=0.002). This preliminary study suggest that telepathology by Internet electronic mail can be a valuable tool for remote consultation in dermatopathology, as well as for other diagnostic fields where expert consultation is necessary.

Novel methods for the determination of the angiogenic activity of human tumors

SummaryAt present the most used method to quantify tumor angiogenesis in human solid tumors is the count of intratumoral microvessels in the primary lesion. This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microvessels. Several studies have found that intratumoral microvessel density (IMD) determined in the primary tumor is significantly associated with metastasis and prognosis in some solid neoplasia, particularly in operable breast carcinoma.The subjective evaluation of IMD made by two observers at the microscope is rapid and of low cost, but presents some difficulties, mainly the identification of the most vascularized area (“hot-spot”) within each tumor. This method can be improved upon to attain a better reproducibility among different pathologists. For example, the use of a multiparametric computerized image analysis system (CIAS) seems to be a promising tool to improve accuracy, feasibility and reproducibility of microvessel counts, although there are still some open technical problems to completely automate its use.Angiogenic activity is the result of a balance between angiogenic stimuli and angio-inhibition. Therefore the determination of angiogenic peptides and/or natural angiogenesis inhibitors in the tumor tissue, serum, or urine of cancer patients seems to be a promising alternative to microvessel counting. At present it is possible to determine the expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and transforming growth factor beta using immunohistochemical methods. Serum and urine levels of bFGF can be assessed using an immunoenzymatic assay. Methods used to assess the expression and levels of urokinase-type plasminogen activator (uPA) or plasminogen activator inhibitor-1 (PAI-1) have also been developed, and correlate with angiogenic activity and prognosis of patients with breast cancer.Finally, some investigational methods to assess angiogenesisin vivo are presented and discussed.Angiogenesis is a very complex phenomenon. Thus it seems reasonable to hypothesize that its assessment by using concurrently several of the available methods may provide more valid, accurate, and comprehensive information on the angiogenic activity of each single tumor. For a reliable and reproducible assessment of angiogenesis for all of the assays, validation procedures and quality control protocols are mandatory.

Robotic Telepathology for Intraoperative Remote Diagnosis Using a Still-Imaging-Based System

The aim of the present study was to assess whether a telemicroscopy system based on static imaging could provide a remote intraoperative frozen section service. Three pathologists evaluated 70 consecutive frozen section cases (for a total of 210 diagnoses) using a static telemicroscopy system (STeMiSy) and light microscopy (LM). STeMiSy uses a robotic microscope, enabling full remote control by consultant pathologists in a near real-time manner. Clinically important concordance between STeMiSy and LM was 98.6% (95.2% overall concordance), indicating very good agreement. The rates of deferred diagnoses given by STeMiSy and LM were comparable (11.0% and 9.5%, respectively). Compared with the consensus diagnosis, the diagnostic accuracy of STeMiSy and LM was 95.2% and 96.2%. The mean viewing time per slide was 3.6 minutes, and the overall time to make a diagnosis by STeMiSy was 6.2 minutes, conforming to intraoperative practice requirements. Our study demonstrates that a static imaging active telepathology system is comparable to dynamic telepathology systems and can provide a routine frozen section service.

Telepathology using Internet multimedia electronic mail: remote consultation on gastrointestinal pathology

The feasibility of using the Internet for remote pathology consultation was examined. We assessed the diagnostic agreement between two groups of pathologists who independently evaluated histopathological cases exchanged by Internet electronic mail. The exchange was between two different workstations using readily available software not specifically developed for telemedicine. Data and images from 76 cases were transmitted to four pathologists. An average of 4.5 images per case were transmitted at compression ratios of between 6:1 and 40:1, corresponding to 250 kByte of data per case. In two cases the remote pathologists could not make a diagnosis. Agreement was reached in 63 of the other 74 (kappa=0.79) =. In 11 cases (15%) there was a misdiagnosis. However, the results are encouraging and suggest that Internet electronic mail can be used successfully for remote consultation in pathology.

Clinical validation of an automated system for supporting the early diagnosis of melanoma

Background: Early diagnosis and surgical excision is the most effective treatment of melanoma. Well‐trained dermatologists reach a high level of diagnostic accuracy with good sensitivity and specificity. Their performances increase using some technical aids as digital epiluminescence microscopy. Several studies describe the development of computerized systems whose aim is supporting dermatologists in the early diagnosis of melanoma. In many cases, the performances of those systems were comparable to those of dermatologists. However, this cannot tell us whether a system is able to support dermatologists. Actually, the computerized system might correctly recognize the same lesions that the dermatologist does, without providing them any useful advice and therefore being useless in recognizing early malignant lesions.

Integrating mHealth in Oncology: Experience in the Province of Trento.

Background The potential benefits of the introduction of electronic and mobile health (mHealth) information technologies, to support the safe delivery of intravenous chemotherapy or oral anticancer therapies, could be exponential in the context of a highly integrated computerized system. Objective Here we describe a safe therapy mobile (STM) system for the safe delivery of intravenous chemotherapy, and a home monitoring system for monitoring and managing toxicity and improving adherence in patients receiving oral anticancer therapies at home. Methods The STM system is fully integrated with the electronic oncological patient record. After the prescription of chemotherapy, specific barcodes are automatically associated with the patient and each drug, and a bedside barcode reader checks the patient, nurse, infusion bag, and drug sequence in order to trace the entire administration process, which is then entered in the patient’s record. The usability and acceptability of the system was investigated by means of a modified questionnaire administered to nurses. The home monitoring system consists of a mobile phone or tablet diary app, which allows patients to record their state of health, the medications taken, their side effects, and a Web dashboard that allows health professionals to check the patient data and monitor toxicity and treatment adherence. A built-in rule-based alarm module notifies health care professionals of critical conditions. Initially developed for chronic patients, the system has been subsequently customized in order to monitor home treatments with capecitabine or sunitinib in cancer patients (Onco-TreC). Results The STM system never failed to match the patient/nurse/drug sequence association correctly, and proved to be accurate and reliable in tracing and recording the entire administration process. The questionnaires revealed that the users were generally satisfied and had a positive perception of the system’s usefulness and ease of use, and the quality of their working lives. The pilot studies with the home monitoring system with 43 chronic patients have shown that the approach is reliable and useful for clinicians and patients, but it is also necessary to pay attention to the expectations that mHealth solutions may raise in users. The Onco-TreC version has been successfully laboratory tested, and is now ready for validation. Conclusions The STM and Onco-TreC systems are fully integrated with our complex and composite information system, which guarantees privacy, security, interoperability, and real-time communications between patients and health professionals. They need to be validated in order to confirm their positive contribution to the safer administration of anticancer drugs.

Design and initial implementation of a regional tele-oncology project.

Two tele-oncology projects have been in progress since 1997 in the Province of Trento in north-east Italy. The common aim of the projects concerns the design and the implementation of a non-surgical tele-oncology system intended to provide a flexible computing environment for the joint management of oncology patients in a wide-area network. The two projects involve both hospital specialists and general practitioners treating oncological patients. The first phase of the project involves the design and implementation of the oncology teleconsultation service.

Exploiting Classifier Combination for Early Melanoma Diagnosis Support

Melanoma is the most dangerous skin cancer and early diagnosis is the main factor for its successful treatment. Experienced dermatologists with specific training make the diagnosis by clinical inspection and they reach 80% level of both sensitivity and specificity. In this paper, we present a multiclassifiers system for supporting the early diagnosis of melanoma. The system acquires a digital image of the skin lesion and extracts a set of geometric and colorimetric features. The diagnosis is performed on the vector of features by integrating with a voting schema the diagnostic outputs of three different classifiers: discriminant analysis, k-nearest neighbor and decision tree. The system is build and validated on a set of 152 skin images acquired via D-ELM. The results are comparable or better of the diagnostic response of a group of expert dermatologists.