Novel chemiluminescent Western blot blocking and antibody incubation solution for enhanced antibody-antigen interaction and increased specificity.

Western blotting is a ubiquitous tool used in protein and molecular biology research, providing information about the presence, size, relative abundance, and state of a protein in a mixture. First, the proteins in a sample are separated by size using SDS-PAGE then transferred onto a membrane for detection with a set of primary and secondary antibodies. High-quality Western data requires high signal-to-noise ratios, which depend upon reduction of nonspecific antibody interactions. Blocking is a critical step in the Western blot method as it prevents the antibodies from binding nonspecifically to the membrane and irrelevant proteins. A solution of nonfat dry milk (NFDM) in physiological buffer is commonly used for this purpose, but does not perform well with every type of antibody and is not optimal for low-abundance proteins. We present a novel blocking solution for chemiluminescent Western blots, AdvanBlock™-chemi, which outperforms NFDM in experiments with 20 unique antibodies by increasing signal-to-noise ratios and minimizing nonspecific binding. This solution enhances protein detection by Western blot and provides consistent results for detection of low abundant and modified proteins.

Child maltreatment: a review of key literature in 2013.

PURPOSES OF REVIEW: This review summarizes new findings in the field of maltreatment, addressing epidemiology, physical abuse, abusive head trauma, sexual abuse, sequelae, and prevention. RECENT FINDINGS: Many articles this year focus on establishing a framework for thinking about how to evaluate a child for maltreatment, the consequences of maltreatment, and the current understanding of prevention efforts. Interestingly, some research has helped to reinforce some concepts that were clinically appreciated, especially related to retinal hemorrhages. SUMMARY: The volume, quality, and breadth of research relating to child maltreatment continue to improve and expand our understanding of child abuse pediatrics. These authors summarize notable advances in our understanding of child maltreatment over the past year.

Cutaneous Findings Mistaken for Physical Abuse: Present but Not Pervasive.

Incorrect diagnoses during child abuse evaluations are serious. Because skin lesions are common in abuse, it is important to consider cutaneous mimics of physical abuse. The current study prospectively identified cutaneous mimics in a cohort of children evaluated for possible physical abuse. This is a secondary analysis of data from the Examining Siblings To Recognize Abuse research network's prospective, observational, cross-sectional study involving 20 U.S. child abuse teams. Subjects were younger than 10 years old and were evaluated by child abuse physicians (CAPs) for concerns of physical abuse. CAPs prospectively documented whether mimics were identified during their physical abuse evaluations. Details of each patient with cutaneous mimics were evaluated to determine the types of mimics, which part of the evaluations identified mimics, and the perceived abuse likelihood. Of 2,890 children evaluated for physical abuse, 137 had at least one mimic identified and 69 had some cutaneous mimic components. Although 985 of 2,753 (39%) subjects without mimics had high levels of abuse concern, only 9 of 137 (6%) children with mimics had high levels of abuse concern (p < 0.001). Of 69 children with cutaneous mimics, 56 (81%) were diagnosed by history and physical examination. Cutaneous abuse mimics were identified in 2.4% of children evaluated for physical abuse. Although it was eventually determined that there was little or no concern for abuse in 84% of children with cutaneous mimics, a small number were physically abused. CAP evaluation may be valuable in recognizing children with cutaneous mimics who also were abused.

Robotic totally endoscopic triple coronary artery bypass grafting on the arrested heart: report of the first successful clinical case.

Robotic technology enables "port only" totally endoscopic coronary artery bypass grafting (TECAB). During early procedure development only single bypass grafts were feasible. Because current referral practice for coronary bypass surgery mostly includes multivessel disease, performance of multiple endoscopic bypass grafts is desirable. We report a case in which a patient received a right internal mammary artery bypass graft to the left anterior descending artery and a left internal mammary artery jump graft to 2 obtuse marginal branches. The procedure was performed through 5 ports on the arrested heart using the daVinci S robotic surgical system. This is the first reported triple bypass grafting procedure using an arrested heart approach.

Non-cutaneous conditions clinicians might mistake for abuse.

OBJECTIVE: To determine the frequency of non-cutaneous mimics identified in a large, multicentre cohort of children evaluated for physical abuse. METHODS: Prospectively planned, secondary analysis of 2890 physical abuse consultations from the Examining Siblings To Recognize Abuse (ExSTRA) research network. Data for each enrolled subject were entered at the child abuse physician's diagnostic disposition. Physicians prospectively documented whether or not a 'mimic' was identified and the perceived likelihood of abuse. Mimics were divided into 3 categories: (1) strictly cutaneous mimics, (2) strictly non-cutaneous mimics and (3) cutaneous and non-cutaneous mimics. Perceived likelihood of abuse was described for each child on a 7-point scale (7=definite abuse). RESULTS: Among 2890 children who were evaluated for physical abuse, 137 (4.7%) had mimics identified; 81 mimics (59.1% of mimics and 2.8% of the whole cohort) included non-cutaneous components. Six subjects (7.4%) were assigned a high level of abuse concern and 17 (20.1%) an intermediate level despite the identification of a mimic. Among the identified mimics, 28% were classified as metabolic bone disease, 20% haematologic/vascular, 16% infectious, 10% skeletal dysplasia, 9% neurologic, 5% oncologic, 2% gastrointestinal and 10% other. Osteomalacia/osteoporosis was the most common non-cutaneous mimic followed by vitamin D deficiency. CONCLUSIONS: A wide variety of mimics exist affecting most disease categories. Paediatric care providers need to be familiar with these conditions to avoid pitfalls in the diagnosis of physical abuse. Identification of a mimic does not exclude concurrent abuse.

Child abuse physicians: coping with challenges.

This article explores how child abuse physicians (CAPs) experience the unique challenges of the emerging field of child abuse pediatrics. Practicing CAPs completed a written survey about known challenges in their field. Fifty-six CAPs completed the written survey and reported experiencing many negative consequences including: threats to their personal safety (52%), formal complaints to supervisors (50%) and licensing bodies (13%), negative stories in the media (23%), and malpractice suits (16%). A purposeful sample of CAPs participated in telephone interviews about these challenges. The 19 physicians who were interviewed described the challenges, while they spontaneously expressed satisfaction with their career and described some strategies for coping with the stresses of child abuse pediatrics. The findings highlight the stressors and challenges that may affect the ability to maintain an adequate CAP workforce. Better understanding of the challenges should help prepare physicians to practice this subspecialty.

Attitudes and beliefs of adolescents and parents regarding adolescent suicide.

OBJECTIVE: The goal was to understand the attitudes, beliefs, and perceptions of adolescents and parents of adolescents, from a variety of backgrounds, regarding adolescent suicide. METHODS: This qualitative study used focus groups to elicit the thoughts of distinct sociodemographic groups. A professional moderator guided the sessions by using a semistructured script. All groups were audiotaped. The transcripts and transcript summaries were analyzed for recurrent themes. The study was performed in community centers and schools in Chicago, Illinois (urban), and the Kansas City, Kansas, area (suburban and rural). A total of 66 adolescents (13-18 years of age) and 30 parents of adolescents participated in 13 focus groups. RESULTS: Both adolescents and parents recognized adolescent suicide as a major problem, but not for their own communities. All parent and adolescent groups identified many risk factors for suicide. Most adolescents reported drug and alcohol use as risk factors for suicide. However, parents often viewed drug and alcohol use as normal adolescent behavior. Both adolescent and parent groups suggested securing or removing guns if an adolescent was known to be suicidal. All participants requested information about adolescent suicide. CONCLUSIONS: Adolescents and parents need help understanding that suicide is an underidentified problem in their own communities. Both adolescents and parents are interested in learning more about how to identify and to intervene with a suicidal adolescent. Pediatricians are well positioned to provide this information in the office and in the community.

Optical coherence tomography imaging as a quality assurance tool for evaluating endoscopic harvest of the radial artery.

BACKGROUND: Concerns about intimal disruption and spasm have limited enthusiasm for endoscopic radial artery harvest (ERAH), although the risk of these problems after this procedure remains uncertain. Radial artery conduits were screened intraoperatively before and after ERAH vs open harvest using catheter-based high-resolution optical coherence tomography (OCT) imaging. METHODS: Twenty-four cadavers and 60 coronary artery bypass graft (CABG) patients scheduled to receive a RA graft underwent OCT imaging before (in situ) and after (ex vivo) open harvest or ERAH. Spasm was quantified by the percentage change in luminal volume between images. Intimal disruption was classified as minor or severe depending on whether the defect was confined to branch ostia or involved the luminal surface. Histology was used to confirm OCT findings. RESULTS: Luminal volume significantly declined after harvest in all RAs from CABG patients, but there was no difference between groups: -43% +/- 29% vs -35% +/- 38% change after ERAH (n = 21) vs open harvest (n = 39; p = 0.342). Significantly more intimal injury was noted after ERAH vs open harvest (34/41 vs 9/43, intimal tears/total evaluated RAs, p < 0.0001). Most intimal injury was minor: only 2 tears involved the luminal surface of the RA (both after ERAH). Serial imaging in cadavers revealed that 86% of ostial tears occur in ERAH during the initial blunt dissection step using the endoscope. CONCLUSIONS: Although branch injury is a pitfall of ERAH, OCT imaging documented that the quality of RA procured is acceptable and comparable with open harvest. Catheter-based OCT provides an important quality assurance tool for RA harvest.

Catheter-based infrared light scanner as a tool to assess conduit quality in coronary artery bypass surgery.

OBJECTIVES: Endothelial disruption within saphenous vein and radial artery grafts increases thrombosis risk. However, no clinically applicable method for imaging the intima currently exists. We used a novel infrared imaging technology, optical coherence tomography (OCT; LightLab Imaging, Inc, Westford, Mass), to visualize the intima within harvested conduits. METHODS: Conduits were procured endoscopically (37 saphenous vein grafts and 8 radial artery grafts) or with the open technique (9 radial artery grafts) from 50 patients. Surplus segments were analyzed by means of OCT for evidence of preexisting pathology or traumatic injury. Focal plaques in radial artery grafts and the intimal hyperplasia area in saphenous vein grafts were quantified as having an intimal/medial thickness ratio of greater than 0.5. Biopsy specimens were obtained for histologic confirmation and to analyze matrix metalloproteinase 2 levels (saphenous vein grafts) and prostacyclin/nitric oxide metabolites (radial artery grafts). Interobserver kappa coefficients and a Bland-Altman analysis were used to determine the reproducibility and accuracy of OCT interpretations. RESULTS: Radial artery imaging revealed plaque in 76%. Endoscopically harvested vessels showed intraluminal clot (38%) and intimal tears ranging from severe (6%) to mild (88%). In saphenous vein grafts intimal thickening was detected in 86% and intraluminal clotting in 68%. The intimal/medial thickness ratio determined by means of OCT correlated directly with matrix metalloproteinase 2 levels (R = 0.6804) in saphenous vein grafts and inversely with metabolites of prostacyclin (R = -0.55) and nitric oxide (R = -0.58) in radial artery grafts. OCT imaging was reproducible (interobserver kappa coefficients of >0.81 for the characterization of plaque types) and showed a strong correlation with histology (R = 0.8, P < .001). CONCLUSIONS: OCT imaging provides an accurate, real-time, and reproducible means for assessing saphenous vein graft and radial artery graft bypass conduits. As a quality assurance tool, this technology might afford a more objective basis for conduit selection.

Strategies to reduce intraluminal clot formation in endoscopically harvested saphenous veins.

OBJECTIVE: Residual clot strands within the excised saphenous vein are an increasingly recognized sequela of endoscopic vein harvest. We hypothesized that endoscopic visualization facilitated by sealed carbon dioxide insufflation causes stagnation of blood within the saphenous vein. In the absence of prior heparin administration, this stasis provokes clot formation. METHODS: Forty consecutive patients having coronary artery bypass grafting underwent endoscopic vein harvest using sealed (Guidant VasoView, n = 30; Guidant Corp, Minneapolis, Minn) or open (Datascope ClearGlide, n = 10; Datascope Corp, Montvale, NJ) carbon dioxide insufflation followed by ex vivo assessment of intraluminal saphenous vein clot via optical coherence tomography. In the sealed carbon dioxide insufflation groups, clot formation was compared with (preheparinized, n = 20) and without (control, n = 10) heparin administration before endoscopic vein harvest, either at a fixed dose or titrated to an activated clotting time greater than 300 seconds. Risk factors for clot formation were assessed. RESULTS: Residual saphenous vein clot was a universal finding in control veins (sealed carbon dioxide insufflation endoscopic vein harvest without preheparinization). At either dose used, heparin given before endoscopic vein harvest significantly decreased saphenous vein clot burden. A similar reduction in clot was observed when using open carbon dioxide insufflation endoscopic vein harvest without preheparinization. Intraoperative blood loss and blood product requirements were similar in all groups. Patient age and preoperative maximum amplitude of the thrombelastography tracing showed a linear correlation with saphenous vein clot volume. CONCLUSION: By enabling the quantification of this issue as never before possible, optical coherence tomography screening revealed that intraluminal saphenous vein clot is frequently found after endoscopic vein harvest. Systemic heparinization before harvest or an open carbon dioxide endoscopic vein harvest system are benign changes in practice that can significantly lessen this complication.

Incidence of residual clot strands in saphenous vein grafts after endoscopic harvest.

OBJECTIVE: : Strands of clot are frequently flushed out of saphenous vein grafts (SVG) during preparation for grafting, particularly those that are endoscopically harvested. However, saline distention at uncontrolled pressures increases graft thrombogenicity and the risk of early failure after coronary artery bypass grafting. The purpose of this prospective investigation was to define the incidence of intraluminal clot within endoscopically harvested SVG and the effect of attempted removal by saline distention. METHODS: : Endoscopically harvested SVG were intraoperatively prepared for grafting by using saline distention at uncontrolled pressure (n = 24) or without distension (n = 20). Optical coherence tomography, a catheter-based infrared imaging system, was used to identify and characterize intraluminal clot strands in surplus SVG segments (average length for analysis, 4.9 ± 2.6 cm). These segments were also assessed for luminal tissue factor activity and percent endothelial integrity by CD31-directed immunohistochemistry. RESULTS: : Clot strands were observed in 45.4% (20 of 44) of imaged SVG segments (severity of observed clots: 54%, mild; 32%, moderate; 14%, severe). Compared with grafts distended with saline, vein segments that were not distended displayed significantly higher endothelial integrity (60.1% ± 27.2% versus 24.7% ± 24.1%, P < 0.05) and lower tissue factor activity (1.28 ± 0.95 versus12.3 ± 5.5 U/cm, P < 0.001) despite having a higher incidence of clot stands (65.0% versus 29.1%, P < 0.02, Fisher exact test). Static flow was observed in veins during endoscopic harvest. CONCLUSIONS: : Clot strands of varying severity are a common finding after endoscopic vein harvest. Saline distention is not completely effective in removing clot strands and increases overall graft thrombogenicity. Therefore, prevention of clot or less traumatic methods of removing clot are indicated.

Off-pump versus on-pump coronary artery bypass grafting in consecutive patients: decision-making algorithm and outcomes.

BACKGROUND: Debate continues between on-pump or off-pump approach for coronary artery bypass grafting (CABG). We used off-pump coronary artery bypass grafting (OPCAB) as a tool within a decision-making algorithm driven by the patient-related factors of coronary anatomy and comorbidity. Our analysis presents this decision algorithm and describes outcomes using this approach. METHODS: From January 2000 to December 2003, 592 consecutive patients undergoing isolated CABG were assigned by one surgeon to a technique: on-pump CABG or OPCAB according to (1) anatomy and (2) predicted risk. Anatomic factors against OPCAB were target vessel size less than 1.25 mm, calcification, poor quality, intramyocardial location, and multiple stenoses. Given that OPCAB could be performed safely, patients in the moderate risk range, ie, those elderly with multiple comorbidities, were preferentially treated using OPCAB. RESULTS: The OPCAB group had higher predicted 30-day mortality compared with the on-pump CABG group, consistent with the protocol's intent. However, morbidity and mortality were similar between on-pump CABG and OPCAB. The OPCAB patients received the same number of internal mammary artery grafts but fewer distal grafts. Mortality and observed to expected ratios were favorable for both groups and below those The Society of Thoracic Surgeons' predicted for OPCAB. CONCLUSIONS: Matching surgical strategy to patient-related factors and needs resulted in excellent outcomes. Our data support the use of a protocol based on patient characteristics to drive the surgeon's choice between an on-pump CABG or OPCAB approach. As such, OPCAB can be viewed as a tool to be used by the surgeon developing a best practice in treating coronary artery disease.

A multi-institutional experience with the CryoMaze procedure.

BACKGROUND: The Cox Maze III operation remains the gold standard for the surgical treatment of atrial fibrillation (AF). We report our experience using a flexible argon-powered metal cryoprobe for creation of the complete Cox-Maze III lesion set in patients with AF (the CryoMaze operation). METHODS: Beginning July 2002, 38 patients underwent the CryoMaze operation. Two patients had isolated CryoMaze procedures, whereas 36 patients had the CryoMaze performed in combination with coronary, valvular, or aortic surgery. All patients had electrocardiographic follow-up. Atrial fibrillation was continuous in 79%. Mean duration of AF before operation was 48 months. RESULTS: There were no complications related to the use of cryoenergy. There was 1 hospital death (2.6%) and no late deaths. At a mean follow-up of 12 months, 35 of 37 patients (95%) were in normal sinus rhythm. One patient required pacemaker implantation 3 months after surgery, and 1 patient refused follow-up. There have been no early or late thromboembolic events. CONCLUSIONS: The CryoMaze operation reliably cures atrial fibrillation at medium term follow-up, with rates identical to those reported for the classic Cox-Maze III operation.