Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide.

Fluorescent in situ hybridisation (FISH) is a powerful tool for the evaluation of chromosomal alterations in formalin fixed paraffin wax embedded sections of colorectal cancer. However, initial experiments using a two-step detection system for digoxigenin labelled chromosome specific centromeric probes resulted in a complete lack of hybridisation signal from a number of colorectal tumour sections. This was due to high levels of background autofluorescence observed in this tissue, which masked any relatively weak hybridisations present. To overcome this problem, a biotinylated tyramide mediated amplification system was incorporated into the FISH detection protocol. This involves the use of horseradish peroxidase to activate the biotinylated tyramide, resulting in the deposition of a large number of biotin molecules at the site of bound peroxidase, which corresponds directly to the location of hybridised probe. Final detection was by means of a streptavidin-FITC conjugate. Using this technique, a panel of 11 colorectal tumour samples studied to date have shown strong, specific hybridisation signals to the nucleus of tumour cells. Amplification of FISH signals by biotinylated tyramide has the potential to improve weak hybridisation signals in cells from numerous sources, using a variety of probe types, including single copy gene probes as well as centromere specific probes.     

Performance on the Balance Error Scoring System Decreases After Fatigue

OBJECTIVE: To determine the immediate effects of a whole-body fatigue protocol on performance of the Balance Error Scoring System (BESS), a postural-stability test commonly used as part of a concussion-assessment battery. DESIGN AND SETTING: Subjects were assigned to a fatigue or control group and were assessed before and immediately after a 20-minute fatigue protocol or rest period. SUBJECTS: Fourteen fatigue subjects and 13 control subjects participated in this study. All subjects were male and free of vestibular disorders, and none had suffered a mild head injury or lower extremity injury in the preceding 6 months, as described through self-report. MEASUREMENTS: We measured performance on the BESS for 9 stance-surface conditions and summed each condition to obtain a total score. Using the Borg scale, we also measured ratings of perceived exertion before, during, and after the fatigue protocol or rest period. RESULTS: We found a significant increase in total errors from pretest to posttest in the fatigue group (14.36 +/- 4.73 versus 16.93 +/- 4.32), a significant decrease in errors in the control group (13.32 +/- 3.77 versus 11.08 +/- 3.88), and a significant difference between groups on the posttest. The rating of perceived exertion scores were significantly different between the fatigue and control groups at the middle (13.29 +/- 1.59 versus 6.23 +/- 0.83) and end (15.86 +/- 2.38 versus 6.15 +/- 0.55) of the fatigue or rest period. CONCLUSIONS: The BESS error scores increased immediately after the fatigue protocol, demonstrating that balance ability diminished. Clinicians who use the BESS as part of their sideline assessment for concussion should not administer the test immediately after a concussion due to the effects of fatigue.     

Simple method for production of internal control DNA for Mycobacterium tuberculosis polymerase chain reaction assays.

A simple method for the production of internal control DNA for two well-established Mycobacterium tuberculosis polymerase chain reaction assays is described. The internal controls were produced from Mycobacterium kansasii DNA with the same primers but at a lower annealing temperature than that used in the standard assays. In both assays, therefore, the internal control DNA has the same primer-binding sequences at the target DNA. One-microgram quantities of internal control DNA which was not contaminated with target DNA could easily be produced by this method. The inclusion of the internal control in the reaction mixture did not affect the efficiency of amplification of the target DNA. The method is simple and rapid and should be adaptable to most M. tuberculosis polymerase chain reaction assays.     

Induction of biologically active antineutrophil cytoplasmic antibodies by immunization with human apoptotic polymorphonuclear leukocytes

Translocation of intracellular components to the cell surface during the priming or apoptosis of polymorphonuclear leukocytes (PMN) is an important mechanism for interaction of antineutrophil cytoplasmic antibodies (ANCA) with these antigens. To test the capacity of apoptotic PMN to trigger production of ANCA, six groups of mice were immunized with either live or apoptotic lymphocytes, or with live, apoptotic, formalin-fixed, or lysed PMN. Mice immunized with both live and apoptotic neutrophils developed high titers of antibodies which gave a granular cytoplasmic immunofluorescent pattern. These antibodies were specific for lactoferrin and myeloperoxidase. Following a second intravenous infusion of apoptotic PMNs, mice developed anti-PR3 antibodies. Vasculitis lesions were not found in mice which developed ANCA. The ANCA-containing IgG fraction induced superoxide production by human PMNs. These results support the hypothesis that neutrophil-specific antigens presented on the cell membranes of apoptotic PMN may induce ANCA in the proper conditions.     

The Prediction of Intracranial Injury After Minor Head Trauma in the Pediatric Population

Reference: Dunning J, Batchelor J, Stratford-Smith P, et al. A meta-analysis of variables that predict significant intracranial injury in minor head trauma. Arch Dis Child. 2004;89:653–659.Clinical Question: Which clinical signs or symptoms of minor head trauma are predictive of intracranial hemorrhage in children and adolescents?Data Sources: Investigations were identified by MEDLINE and EMBASE searches from 1990 through 2002 by a search of the grey literature and by contacting experts for additional papers. The search terms were selected to find all studies reporting intracranial hemorrhage (ICH) or complications after head trauma.Study Selection: A full systematic review was conducted, and all cohort or nested cohort studies that presented data on minor head injuries in children less than 18 years old, with or without ICH, were identified. Studies were then judged for inclusion based on the presentation of a series of at least 100 patients and a documented reliable standard for the detection of ICH for all patients in the study. The use of computed tomography (CT) and medical follow-up was considered an acceptable gold standard. Intracranial hemorrhage was defined as any abnormality detected on the CT scan due to the traumatic presence of extravascular blood. Minor head trauma was defined as patients presenting with a Glasgow Coma Scale (GCS) score of 13–15.Data Extraction: Seven clinical correlates were used for data extraction, including skull fracture, headache, vomiting, focal neurology, seizure, loss of consciousness, and a GCS score of less than 15. Data were analyzed using a pooled estimate of the relative risk ratio with a random-effects model.Main Results: The searches identified a total of 2134 studies for the initial review. After an abstract review by 2 independent examiners, 98 studies were identified for a full-paper review. Each study was graded on a 4-point scale according to the level of evidence provided, using scales consistent with the Oxford Centre for Evidence-Based Medicine and the National Institute for Clinical Excellence. Thirty-four of these articles were of adequate quality for inclusion; however, many did not include data that could be separated into a specific data set for children, had too small a sample size, or lacked enough data on individual correlates to head trauma. Nineteen studies provided data on children, but 3 of these were excluded due to poor quality or lack of a reported CT scan, leaving a total of 16 studies for the meta-analysis.The analysis included a total of 22 420 patients ranging between 0 and 18 years of age. The meta-analysis showed a significant increased relative risk of ICH for patients sustaining loss of consciousness (2.23), GCS <15 (5.51), skull fracture (6.13), and focal neurology (9.43). No significant increases in risk for headache (1.02), vomiting (0.878), or seizure (2.82) were noted; however, heterogeneity was significant for this last correlate. The prevalence of ICH ranged from 1.3 to 36%, supporting the notion of a large amount of heterogeneity or variability in the inclusion criteria among the studies.Conclusions: These findings demonstrate that loss of consciousness, decreased level of consciousness (GCS <15), skull fracture, and focal neurology are risk factors for ICH in the pediatric population. However, these findings are not definitive enough to establish pediatric head-injury guidelines regarding CT scanning or admission to hospital after minor head trauma.COMMENTARYAlthough intracranial hemorrhage (ICH) after mild head injury is a rare occurrence in athletes, certified athletic trainers (ATCs) must be aware of the signs and symptoms of all severities of head trauma, including ICH. The initial role of the ATC when there is a suspected head injury is the detection of focal traumatic brain injury (TBI), including epidural hematoma, subdural hematoma, cerebral contusion, and intracerebral hemorrhage and hematoma.^1,2 To successfully recognize these potentially life-threatening head injuries, the ATC must understand the various presentations of athletes with head injuries and the signs and symptoms that often accompany them, such as loss of consciousness (LOC), cranial nerve deficits, decreasing mental status, and worsening symptoms.¹Dunning et al³ presented a meta-analysis that has direct relevance to the practice of athletic training and the management of minor head injuries. Understanding the potential risk factors for ICH is an important step in ensuring adequate referral to medical professionals and a quick diagnosis of possible ICH. Often, the ATC must decide whether an athlete should be referred to the emergency room once he or she has sustained a mild head injury and, once at the emergency room, physicians need to decide on a course of diagnostics. This is even more of a concern in the pediatric athlete because of the potential for both short-term and long-term complications in the still-developing brain.^4–7 As a general rule, failure of an athlete''s mental status to clear rapidly should lead to a referral for neuroimaging.⁸ With the suspicion of focal TBI, CT scans have been recommended as the neuroimaging modality of choice because they can easily detect acute blood collection and skull fracture.⁸It is important to note that differences exist between sport-related minor head trauma and minor head trauma from additional mechanisms, such as motor vehicle accidents, falls, and other accidents. None of the studies used by Dunning et al³ were investigations of sport-related minor head injury. Minor head injuries that produce ICH or any of the clinical correlates found to be significant predictors of ICH (LOC, focal neurology, Glasgow Coma Scale [GCS] <15, or skull fracture) are rare in athletes.² In fact, recent authors have reported that only 6.3 to 8.9% of collegiate athletes experienced LOC after a concussion.^9–11 Regardless of the rarity of focal TBI and injuries that result in ICH during athletics, it is imperative that these injuries be ruled out by the ATC.Based on this meta-analysis, the correlates identified as predictors of ICH included LOC, a GCS score of <15, focal neurology, and a skull fracture. Fortunately, the presence of these clinical signs and symptoms in an athlete would warrant physician referral based on the recommendations made in the National Athletic Trainers'' Association position statement on sport-related concussion and other recommendations for on-field management of head trauma.^1,2,8 Even though headache and vomiting were not predictive of ICH, documenting these and other signs and symptoms of mild head trauma should be part of the ATC''s assessment protocol.^1,12,13 By quantifying the number of signs and symptoms present as well as the frequency and/or duration of these signs and symptoms, the ATC can track the recovery of the athlete and use the information for referral if the athlete does not demonstrate improvement. In addition, the ATC should use age-appropriate adjunct assessments, including neurocognitive testing^7,14–18 (traditional pen-and-paper neuropsychological tests, ImPACT [Immediate Postconcussion Assessment and Cognitive Testing, University of Pittsburgh Medical Center, Pittsburgh, PA], ANAM [Automated Neuropsychological Assessment Metrics, National Rehabilitation Hospital Assistive Technology and Neuroscience Center, Washington, DC], Concussion Resolution Index [HeadMinder Inc, New York, NY], Standardized Assessment of Concussion [CNS Inc, Waukesha, WI]) and postural stability testing¹⁸ to aid in the decision-making process.The findings of Dunning et al³ provide insight into specific risk factors the ATC should look for when evaluating minor head trauma in children and adolescents. The presence of any of the significant predictors should warrant immediate referral. The authors also acknowledge that other signs and symptoms (eg, dizziness, drowsiness, confusion) could be predictive of ICH; however, these factors had not been adequately investigated in the pediatric literature identified for this meta-analysis and therefore were not included. This factor, along with the variability in the inclusion criteria, timing of CT scans, and differences in the ICH definitions in the individual studies are limitations of this meta-analysis. Another potential limitation regarding the predictive value of the headache variable to ICH stems from a lack of information regarding the severity of the headaches reported in the individual studies. Some evidence suggests a relationship between severe headaches and ICH¹⁹; therefore, headache severity should also be questioned during the clinical examination. Several other limitations of this meta-analysis include no listing of specific medical subject headings terms used to search the databases, not describing the duration of LOC from the various studies used, and not adequately describing or defining the specific types of focal neurology used as a correlate. However, other authors have described focal neurologic changes as including posturing and dilating pupils.²Although this meta-analysis offers medical professionals working with children and adolescents one interpretation of the evidence regarding clinical risk factors predictive of ICH, it does not provide strong enough evidence to alter the current head-injury management and CT scanning protocols for children.²⁰ Future studies should address the limitations outlined by Dunning et al³ to better determine the predictive value of various clinical signs and symptoms of minor head trauma in the pediatric population.     

Neurobehavioral phenotype in carriers of the fragile X premutation

There have been contradictory findings in the fragile X (fraX) literature about possible neurocognitive and psychological symptoms due to the fraX premutation (pM). The purpose of the present study was to investigate the relationship between CGG repeat length and neurobehavioral functioning in carriers of the fraX pM. Eighty‐five female carriers of the pM with allele sizes ranging from 59–166 were administered a comprehensive IQ test (WAIS‐III) and completed a questionnaire designed to measure psychopathology (Symptom Checklist (SCL)‐90‐R). No relationship between allele size and cognition was identified. A significant negative relationship between allele size and age was found, as well as a positive relationship between allele size and depression. Follow‐up analyses separating small and large allele sizes (below and above 100 CGG repeats) indicated that individuals with larger allele sizes scored significantly higher on the Interpersonal Sensitivity and Depression subscales of the SCL‐90‐R. Despite the limitation of few individuals with high CGG repeat lengths, our findings suggest that females with larger premutated alleles (≥ 100 repeats) display some clinical manifestations of fraX syndrome. © 2001 Wiley‐Liss, Inc.