Jeffrey M. Levsky

Fluorescence in situ hybridization: past, present and future

Fluorescence in situ hybridization (FISH), the assay of choice for localization of specific nucleic acids sequences in native context, is a 20-year-old technology that has developed continuously. Over its maturation, various methodologies and modifications have been introduced to optimize the detection of DNA and RNA. The pervasiveness of this technique is largely because of its wide variety of applications and the relative ease of implementation and performance of in situ studies. Although the basic principles of FISH have remained unchanged, high-sensitivity detection, simultaneous assay of multiple species, and automated data collection and analysis have advanced the field significantly. The introduction of FISH surpassed previously available technology to become a foremost biological assay. Key methodological advances have allowed facile preparation of low-noise hybridization probes, and technological breakthroughs now permit multi-target visualization and quantitative analysis - both factors that have made FISH accessible to all and applicable to any investigation of nucleic acids. In the future, this technique is likely to have significant further impact on live-cell imaging and on medical diagnostics.

Acute Aortic Syndromes: A Second Look at Dual-Phase CT

OBJECTIVE The purpose of this article is to assess the diagnostic performance of the unenhanced and contrast-enhanced phases separately in patients imaged with CT for suspected acute aortic syndromes. MATERIALS AND METHODS All adults (n = 2868) presenting to our emergency department from January 1, 2006, through August 1, 2010, who underwent unenhanced and contrast-enhanced CT of the chest and abdomen for suspected acute aortic syndrome were retrospectively identified. Forty-five patients with acute aortic syndrome and 45 healthy control subjects comprised the study population (55 women; mean age, 61 ± 16 years). Unenhanced followed by contrast-enhanced CT angiography (CTA) images were reviewed. Contrast-enhanced CTA examinations of case patients and control subjects with isolated intramural hematoma were reviewed. Radiation exposure was estimated by CT dose-length product. RESULTS Forty-five patients had one or more CT findings of acute aortic syndrome: aortic dissection (n = 32), intramural hematoma (n = 27), aortic rupture (n = 10), impending rupture (n = 4), and penetrating atherosclerotic ulcer (n = 2). Unenhanced CT was 89% (40/45) sensitive and 100% (45/45) specific for acute aortic syndrome. Unenhanced CT was 94% (17/18) and 71% (10/14) sensitive for type A and type B dissection, respectively (p = 0.142). Contrast-enhanced CTA was 100% (8/8) sensitive for isolated intramural hematoma. Mean radiation effective dose was 43 ± 20 mSv. CONCLUSION Unenhanced CT performed well in detection of acute aortic syndrome treated surgically, although its performance does not support its use in place of contrast-enhanced CTA. Unenhanced CT may be a reasonable first examination for rapid triage when IV contrast is contraindicated. Contrast-enhanced CTA was highly sensitive for intramural hematoma, suggesting that unenhanced imaging may not always be needed. Acute aortic syndrome imaging protocols should be optimized to reduce radiation dose.

Coronary Computed Tomography Angiography Versus Radionuclide Myocardial Perfusion Imaging in Patients With Chest Pain Admitted to Telemetry: A Randomized Trial

BACKGROUND Coronary computed tomography angiography plays an expanding role managing symptomatic patients with suspected coronary artery disease. Prospective intermediate-term outcomes are lacking.

A New, Simple Method for Estimating Pleural Effusion Size on CT Scans

BACKGROUND There is no standardized system to grade pleural effusion size on CT scans. A validated, systematic grading system would improve communication of findings and may help determine the need for imaging guidance for thoracentesis. METHODS CT scans of 34 patients demonstrating a wide range of pleural effusion sizes were measured with a volume segmentation tool and reviewed for qualitative and simple quantitative features related to size. A classification rule was developed using the features that best predicted size and distinguished among small, moderate, and large effusions. Inter-reader agreement for effusion size was assessed on the CT scans for three groups of physicians (radiology residents, pulmonologists, and cardiothoracic radiologists) before and after implementation of the classification rule. RESULTS The CT imaging features found to best classify effusions as small, moderate, or large were anteroposterior (AP) quartile and maximum AP depth measured at the midclavicular line. According to the decision rule, first AP-quartile effusions are small, second AP-quartile effusions are moderate, and third or fourth AP-quartile effusions are large. In borderline cases, AP depth is measured with 3-cm and 10-cm thresholds for the upper limit of small and moderate, respectively. Use of the rule improved interobserver agreement from κ = 0.56 to 0.79 for all physicians, 0.59 to 0.73 for radiology residents, 0.54 to 0.76 for pulmonologists, and 0.74 to 0.85 for cardiothoracic radiologists. CONCLUSIONS A simple, two-step decision rule for sizing pleural effusions on CT scans improves interobserver agreement from moderate to substantial levels.

The spatial order of transcription in mammalian cells.

We have previously developed technology for multiplexing probes for the detection of transcription of many genes simultaneously within single cells. This has allowed us to determine the spatial localization of multiple genes with respect to each other in the nucleus, and ultimately the expression profile of the cell with respect to surrounding cells in a tissue. Six parameters of transcriptional organization in individual cells from culture and tissue were used to characterize significant differences in intracellular and intercellular expression patterns while preserving cellular morphology and histological context. We found that, unlike yeast, mammalian expression is excluded from the periphery and in addition, a subtle but complex organization underlies the transcriptional activity of these cells, both intra‐ and intercellularly. The approach has sufficient spatial resolution to be applied to the detection of chromosomal translocations or the identification of cancer cells. J. Cell. Biochem. 102: 609–617, 2007.

Should CT Play a Greater Role in Preventing the Resection of Granulomas in the Era of PET

OBJECTIVE PET cannot distinguish between bronchogenic carcinoma and granuloma, but positive scans may prompt surgery. We systematically evaluated the CT appearance of resected carcinomas and granulomas to identify features that could be used to reduce granuloma resections. MATERIALS AND METHODS We retrospectively identified 93 consecutive patients between January 2005 and November 2008 who had resection of a pulmonary nodule pathologically diagnosed as bronchogenic carcinoma or granuloma and preoperative imaging with CT and PET. Each nodule was evaluated on CT for size, doubling time, location, borders, shape, internal characteristics, calcification, clustering, air bronchograms, and cavitation. A diagnostic impression was rendered. Bivariate and logistic regression analyses were performed. Pre-PET data regarding the proportion of resected granulomas and carcinomas between January 1995 and December 1996 were reviewed. RESULTS Sixty-eight percent (65/96) of nodules were carcinomas and 32% (31/96) were granulomas. The CT impression was benign in 65% (20/31) of granulomas and 5% (3/65) of carcinomas (p < 0.0001; negative predictive value [NPV], 87% [20/23]). Specific CT features significantly associated with granuloma were clustering, cavitation, irregular shape, lack of pleural tags, and solid attenuation. The combination of nonspiculated borders, irregular shape, and solid attenuation had an NPV of 86% (12/14). Granulomas represented 18% (9/50) of resected nodules in 1995 and 1996 (p = 0.066). CONCLUSION CT findings reduce but cannot eliminate the possibility that a nodule is malignant. Outcomes-based clinical trials are needed to determine whether CT features of benignity can guide less-invasive initial management and reverse a concerning trend in granuloma resection.

Rationale and design of a randomized controlled trial comparing stress myocardial perfusion imaging with coronary CT angiography as the initial imaging study for intermediate-risk patients admitted with chest pain

BACKGROUND Noninvasive cardiac imaging plays an important role in the diagnosis and management of coronary artery disease (CAD). Prior studies have focused on the diagnostic performance of noninvasive modalities using angiographically significant stenoses as the reference standard. Recent trends in evidence-based medicine and increasing imaging utilization call for validation of diagnostic algorithms with well-designed, controlled trials with clinical outcome endpoints. OBJECTIVE To compare the performance of stress radionuclide myocardial perfusion imaging (MPI) and coronary computed tomography angiography (CTA) in terms of outcomes. METHODS We designed a single-center, randomized controlled trial that compares MPI and CTA as the initial modality for the evaluation of patients hospitalized for chest pain without known CAD or acute myocardial infarction. Patients with intermediate-risk characteristics and a clinical need for noninvasive imaging are included. The primary outcome measured is the incidence of conventional angiography not leading to subsequent coronary revascularization within 1 year. The study is powered to detect a reduction from 11% to 3% in catheterization not leading to an intervention with a sample size of 400. Secondary outcomes include procedural complications and posttest renal dysfunction (safety outcomes), major adverse cardiovascular events, length of hospital stay, subsequent hospitalizations and imaging, changes in medical management, and tolerability of the noninvasive test. CONCLUSIONS The results of this trial will further our understanding of the relative appropriateness of CTA and MPI in evaluating intermediate-risk patients hospitalized with chest pain. It will also have implications for the design and probability of success of multicentered trials that are currently being planned.

Normal myocardial perfusion on 64-detector resting cardiac CT

BACKGROUND Computed tomography (CT) of the heart is increasingly used to characterize not only the coronary arteries but also cardiac structure and function. The performance of CT in depicting myocardial perfusion is under active investigation. OBJECTIVE We describe the pattern of normal myocardial perfusion on resting 64-detector cardiac CT. METHODS Patients (n = 33; 20 women, 13 men; mean age, 52 years) with normal radionuclide myocardial perfusion imaging and normal coronary arteries on CT angiography (120 kVp) comprised the study population. Segmental myocardial perfusion on CT was measured in Hounsfield units (HU) with manual and semiautomated methods for the 17-segment American Heart Association model in both systole and diastole. Segments were aggregated into coronary artery territories, from apex to base and by myocardial wall. The relationships between myocardial perfusion and various patient factors were evaluated. RESULTS Overall mean myocardial perfusion was 98 HU in systole and 94 HU in diastole with the manual method (P = .011) and 92 HU in systole and 95 HU in diastole with the automated method (P = .001). The septum showed significantly higher mean attenuation values than the other walls in systole and diastole with both methods. Generally, attenuation values were lower in the left circumflex artery territory and in the apex. Bivariate analysis showed higher mean myocardial attenuation values for women than men, although this difference did not persist on multivariate analysis adjusted for patient size. CONCLUSION Normal mean resting myocardial perfusion correlates with CT attenuation values of approximately 92-98 HU on CT angiography in the coronary arterial phase. The septum consistently shows greater attenuation values than the other walls.

Non-invasive assessment of low risk acute chest pain in the emergency department: A comparative meta-analysis of prospective studies

BACKGROUND The aim of this meta-analysis was to compare the diagnostic accuracy of cardiac computed tomographic angiography (CCTA), stress echocardiography (SE) and radionuclide single photon emission computed tomography (SPECT) for the assessment of chest pain in emergency department (ED) setting. METHODS A systematic review of Medline, Cochrane and Embase was undertaken for prospective clinical studies assessing the diagnostic efficacy of CCTA, SE or SPECT, as compared to intracoronary angiography (ICA) or the later presence of major adverse clinical outcomes (MACE), in patients presenting to the ED with chest pain. Standard approach and bivariate analysis were performed. RESULTS Thirty-seven studies (15 CCTA, 9 SE, 13 SPECT) comprising a total of 7800 patients fulfilled inclusion criteria. The respective weighted mean sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and total diagnostic accuracy for CCTA were: 95%, 99%, 84%, 100% and 99%, for SE were: 84%, 94%, 73%, 96% and 96%, and for SPECT were: 85%, 86%, 57%, 95% and 88%. There was no significant difference between modalities in terms of NPV. Bivariate analysis revealed that CCTA had statistically greater sensitivity, specificity, PPV and overall diagnostic accuracy when compared to SE and SPECT. CONCLUSIONS All three modalities, when employed by an experienced clinician, are highly accurate. Each has its own strengths and limitations making each well suited for different patient groups. CCTA has higher accuracy than SE and SPECT, but it has many drawbacks, most importantly its lack of physiologic data.

Resting Cardiac 64-MDCT Does Not Reliably Detect Myocardial Ischemia Identified by Radionuclide Imaging

OBJECTIVE CT myocardial perfusion imaging is an emerging diagnostic modality that is under intensive study but not yet widely used in clinical practice. The purpose of this study is to evaluate the performance of resting 64-MDCT in revealing ischemia identified on radionuclide myocardial perfusion imaging (MPI). MATERIALS AND METHODS We retrospectively identified 35 patients (20 women and 15 men; mean age, 52 years) with myocardial ischemia found on MPI who underwent retrospectively gated CT within 90 days of MPI. Myocardial perfusion on CT was evaluated using both a visual (n = 35) and an automated (n = 34) method. For the visual method, myocardial segments were evaluated qualitatively in systole and diastole. For the automated method, subendocardial perfusion of the standard 17 American Heart Association segments was measured using a commercially available tool in both systole and diastole. Differences between systolic and diastolic perfusion were computed. RESULTS Five hundred eighty myocardial segments were evaluated, 152 of which were ischemic on MPI. Visual analysis had a sensitivity of 16% (24/152), specificity of 92% (393/428), positive predictive value of 40% (24/60), and negative predictive value of 75% (392/520) in systole, and a sensitivity of 18% (27/152), specificity of 89% (382/428), positive predictive value of 37% (27/73), and negative predictive value of 75% (382/507) in diastole, as compared with MPI. There was no significant difference in subendocardial perfusion between ischemic and nonischemic segments by the automated method. There was no significant difference in CT perfusion between patients with and without obstructive coronary artery disease on CT angiography using the visual or automated methods. CONCLUSION Resting 64-MDCT is unsuitable for clinical use in revealing ischemia seen on MPI.