3D ultrasound for prostate localization in radiation therapy: a comparison with implanted fiducial markers

This study compares prostate localization using three-dimensional ultrasound (3D US) to a standard technique using implanted fiducial markers (FMs) for prostate image guided radiation therapy (IGRT). Two methods to determine prostate position on US were evaluated: Assisted segmentation (prospectively) and manual segmentation (retrospectively). Daily couch shifts to align the prostate into treatment position were measured using each technique. A total of 278 FM couch shifts and 255 and 218 corresponding assisted and manual segmentation US couch shifts were analyzed in each direction: Anterior-posterior, right-left, and superior-inferior. Ninety five percent "limits-of-agreement" (LOA) were used to analyze paired couch shifts and to determine if US can reliably replace FMs. We chose an error tolerance of +/- 3 mm for the LOA analysis. For FM vs assisted-segmentation US, 35.3%, 51.0%, and 48.2% of couch shifts (anterior-posterior, right-left, and superior-inferior, respectively) agreed within +/- 3 mm. Agreement improved using manual segmentation US (corresponding agreements were 45.3%, 64.1%, and 55.2%), however, results still lie markedly below the 95% we consider to indicate clinical equivalence. Based on these results, our experience indicates US cannot replace FMs for prostate IGRT, using either assisted or manual segmentation. US couch shifts showed considerably greater variability than FM measures and US image quality is shown to affect agreement. Planning target volume margins for use with the US system were found to be 15.8, 8.7, and 12.5 mm for assisted segmentation and 13.1, 7.6, and 9.8 mm for manual segmentation. Comparison of these margins to those reported in recent studies for use with FM IGRT indicate FMs offer greater sparing of the rectum and bladder than the US system.     

Morphometry of the kangaroo spine and its comparison with human spinal data

The upright posture of the kangaroo suggests that the spine of the kangaroo could be a possible substitute model for biomechanical studies of the human spine. A prerequisite for this should be the agreement of anatomy in humans and kangaroos. The purpose of this study was to investigate the anatomical parameters of the kangaroo spine from C4 to S4 and compare them with existing anatomical data of the human spine. Eight complete spines of the red giant kangaroo were obtained and 21 anatomical parameters were measured from the vertebral bodies, spinal canal, endplate, pedicles, intervertebral discs, transverse, and spinous processes. Most similarities between kangaroo and human spines were found for the vertebral bodies in the cervical and the lumbar spine. The largest differences were evident for the spinous processes. Although both species are somehow upright, these differences may be explained by the way how they move. Jumping probably requires more muscle strength than walking on two legs.     

HLA-B*5701 typing by sequence-specific amplification: validation and comparison with sequence-based typing

Susceptibility to abacavir hypersensitivity (ABC HSR) is strongly associated with alleles carried on the 57.1 ancestral haplotype including HLA-B*5701 and Hsp70 Hom M493T. In one study, prospective testing for HLA-B*5701 and exclusion of individuals carrying this allele, from receiving abacavir, substantially lowered the incidence of ABC HSR to 0% (95% confidence interval 0-0.075%). The presence of HLA-B*5701 is usually detected by standard serological tests and by molecular genetic methods such as sequence-based typing (SBT). While the former test cannot discriminate between HLA-B57 subtypes, the expensive SBT may not be readily available in all laboratories. Hence, an alternate method was developed to detect HLA-B*5701 using allele and group-specific polymerase chain reaction-sequence-specific primers (PCR-SSP) typing. This PCR-SSP-typing method positively amplified all HLA-B*5701 alleles in concordance with their SBT-assigned typing. This multiplexed SSP assay was able to distinguish between HLA-B*5701 (n = 10) and closely related HLA-B57 alleles B*5702 (n = 2), -B*5703 (n = 1), -B*5704 (n = 1) alleles and non-HLA-B*57 alleles (n = 61). In conclusion, this method of HLA-B*5701 detection is a rapid and accurate typing method with high specificity, sensitivity and reproducibility.     

A comparison between multiple and single pregnancies obtained by in-vitro fertilization

Thirty-eight single and 10 multiple pregnancies obtained afterin-vitro fertilization were compared. In the group of multiplegestations, maternal age was lower and the amounts of ovulatorydrugs given were significantly smaller than in relation to singlepregnancies. All multiple pregnancies arose from triple embryotransfers and the embryos from this group exhibited significantlyhigher vitality scores. In both groups, plasma levels of oestradioland progesterone followed the same pattern until day 8 afteroocyte retrieval. Following implantation, the secretion of thesehormones increased more rapidly in multiple pregnancies pointingat greater luteal activity in this group. HCG levels becamesignificantly higher in multiple gestation on day 25 after oocytecollection. Echographic examination showed that, compared tonormal pregnancy, growth in both groups of IVF conceptuses wasinitially retarded but caught up with normal evolution at {smalltilde}30 days after egg retrieval. The need for adjusting thenumber of embryos transferred not only to expected success ratesbut also to the risk of high rank multiples is emphasized.     

The measurement of fractionated bilirubin by Ektachem film slides. Method validation and comparison of conjugated bilirubin measurements with direct bilirubin in obstructive and hepatocellular jaundice

The authors evaluated the Kodak Ektachem Slides for total, "direct," conjugated, unconjugated, and albumin-bound ("delta") bilirubin. For various concentrations of control material, the precision (CV) within- and between-day ranged from 1.7 to 2.4% (2.8-6.6%) for total bilirubin, 0.8-4.2% (1.6-11.5%) for unconjugated bilirubin, and 1.6-9.5% (2.6-20.7%) for conjugated bilirubin. The Ektachem total and "direct" bilirubin assays demonstrated excellent correlation with the Jendrassik and Grof procedure; a 30% difference was observed, however, between absolute numbers with the two direct bilirubin methods. We found the measurement of true conjugated bilirubin by the Kodak Method to be superior to the measurement of "direct" bilirubin in following the response to treatment of various hepatic disorders manifested by extrahepatic and intrahepatic biliary obstruction.     

Comparison of histometric data obtained by optical coherence tomography and routine histology

There is a lack of systematic investigations comparing optical coherence tomography (OCT) with histology. OCT assessments were performed on the upper back of 16 healthy subjects. Epidermis thickness (ET) was assessed using three methods: first, peak-to-valley analysis of the A-scan (ET-OCT-V); second, manual measurements in the OCT images (ET-OCT-M); third, light microscopic determination using routine histology (ET-Histo). The relationship between the different methods was assessed by means of the Pearson correlation procedure and Bland and Altman plots. We observed a strong correlation between ET-Histo (79.4+/-21.9 microm) and ET-OCT-V (79.2+/-15.5 microm, r=0.77) and ET-OCT-M (82.9+/-15.8 microm, r=0.75), respectively. Bland and Altman plots revealed a bias of -0.19 microm (95% limits of agreement: -27.94 microm to 27.56 microm) for ET-OCT-V versus ET-Histo and a bias of 3.44 microm (95% limits of agreement: -24.9 microm to 31.78 microm) for ET-OCT-M versus ET-Histo. Despite the strong correlation and low bias observed, the 95% limits of agreement demonstrated an unsatisfactory numerical agreement between the two OCT methods and routine histology indicating that these methods cannot be employed interchangeably. Regarding practicability, precision, and indication spectrum, ET-OCT-V and ET-OCT-M are of different clinical value.     

Standardization and automation of HLA B27 typing by flow cytometry: validation and comparison with microlymphocytotoxicity

One of the strongest known association between human leukocyte antigen (HLA) phenotype and disease is that of ankylosing spondylitis and HLA-B27. Thus, the determination of HLA-B27 status is an useful tool in the diagnosis of ankylosing spondylitis. To date, the 2 reference methods for HLA typing (microlymphocytotoxicity and molecular biology techniques), are costly in terms of both technician time and materials, and require a great deal of experience. In total, these techniques are not well-suited for routine application in clinical immunology laboratories. Use of flow cytometry has recently been applied for HLA-B27 typing. Nevertheless, it requires an extensive validation protocol. We developed a flow cytometry technique as standardized as possible (whole blood, automated lysing system, automated photomultiplier voltage calibration, definition of thresholds stable with time) and validated our results by comparison with microlymphocytotoxicity. In total, 326 samples were analyzed. We found 99% of concordant results between the 2 techniques, and neither false positive results nor false negative results with flow cytometry could be observed. These results illustrate the reliability of the protocol. It should be remembered that reference technique remains necessary to confirm the few results (< 1%) found in "grey zone" by flow cytometry. Standardization of flow cytometry techniques, as described in this work for HLA B27, seems to be a reasonable goal for the next decade in clinical immunology laboratories.     

Sleep estimation using wrist actigraphy in adolescents with and without sleep disordered breathing: a comparison of three data modes

INTRODUCTION: Adolescence is a time of rapid changes in sleep habits and rising prevalence of sleepiness. The importance of measuring sleep in this population is increasingly recognized. In adults, measurements of sleep by actigraphy correlate well with sleep data from EEG recordings. Since actigraphy is increasingly utilized in adolescent sleep studies, more information is needed about reliability in this age group. This analysis investigated which actigraphy data mode is optimal for data collection in adolescents and explored the level of agreement between actigraphy and polysomnography (PSG) in population subgroups. METHODS: 181 adolescents aged 12-16 years were concurrently monitored with PSG and wrist actigraphy (measured in 3 data modes: Time Above Threshold [TAT], Zero Crossing Mode [ZCM], and Proportional Integration Mode [PIM]) to measure total sleep time (TST). RESULTS: The sample was 50% male, 55% African American, 9% with sleep disordered breathing (SDB; apnea-hypopnea index > or = 5). Intraclass correlation coefficients (ICC) for TST between actigraphy and PSG were low to moderate and were highest for TAT (0.41) compared to ZCM (0.32) and PIM (0.34). Subgroup analyses revealed that ICCs were higher among those without SDB (0.55) than those with SDB (0.00), and for girls (0.66) compared with boys (0.31). CONCLUSIONS: Results suggest that actigraphy provides a reasonably good estimate of TST in adolescents without SDB. Recognition of the variation in sleep estimates among different data collection modes, among population subgroups, and across the age spectrum, may be of fundamental importance in the interpretation of actigraphy data for sleep duration estimation.     

Nasal polyps in patients with and without cystic fibrosis: a differentiation by innate markers and inflammatory mediators

BACKGROUND: The dysfunction of the mucosal interface of the upper respiratory tract in cystic fibrosis (CF) patients is clinically visible by the development of nasal polyps (NP) at a young age. Innate defence markers and inflammatory mediators in NP from patients with CF were compared with non-cystic fibrosis nasal polyps (non-CF-NP) to determine a possible different immunological background in macroscopically similar tissue. METHODS: Surgical samples were obtained from patients with non-CF-NP, cystic fibrosis patients with nasal polyps (CF-NP) and control patients (CO). With real time PCR, the mRNA expression of human beta defensins (HBD) 2 and 3, toll-like receptors (TLR) 2 and 4 and the macrophage mannose receptor (MMR) were measured. On homogenates of the surgical samples eotaxin, myeloperoxidase (MPO), IL-5 and IL-8 protein content was measured using commercial ELISA kits; IgE and eosinophilic cationic protein (ECP) were measured by the Unicap system. RESULTS: In CF-NP we found a statistically significant higher mRNA expression of HBD 2 compared with non-CF-NP and CO and of TLR 2 compared with non-CF-NP. In the non-CF-NP group, MMR mRNA expression was significantly elevated compared with CO and CF-NP. For TLR 4 mRNA expression no statistically significant differences were found between groups. IL-5 was below detection level in all CO and CF-NP, but was measurable in 80% of the non-CF-NP. MPO and IL-8 concentrations were significantly higher in CF-NP compared with CO and non-CF-NP, whereas ECP, eotaxin and IgE were significantly higher in the non-CF-NP group. CONCLUSIONS: We here demonstrate that CF-NP and non-CF-NP not only differ in terms of inflammatory mediator profile, but also in terms of innate markers.     

A procedure for the measurement of infant skin conductance and its initial validation using clap induced startle

Studies examining psychophysiologic markers of infant emotional development abound. However, few studies have used skin conductance (SC), though it measure's sympathetic activity, and none have measured SC on infants sitting up and actively engaged with another person, a significant challenge given the measures sensitivity to movement artifact. In this pilot/feasibility study, we present a procedure for measuring infant SC during active engagement with another person who executes a series of startling claps to elicit an SC response. We measured SC on the plantar surface of the foot of seventeen 5-month-old infants. We found unconditioned SC responses that were related to the intensity of physical startle reactions for each clap trial. We also found anticipatory, conditioned SC responses that occurred within 5 s before each clap that occurred when the researcher raised his clasped hands. These conditioned SC responses grew linearly in intensity over trials. We conclude that SC may be a useful addition to the infant researcher's armamentarium and may indeed be used to measure physiologic reactivity in infants even when actively engaged with another person. Addition of SC measurement to research on infant emotion and emotional communication is likely to advance our understanding of the psychophysiologic foundations of infant emotional development.     

An estimation of noise levels in HMPAO RCBF SPECT images using simulation and phantom data; comparison with results obtained from repeated normal controls

The purpose of this work was to determine noise levels in HMPAO RCBF SPECT images. Eight simulated images of a uniform sphere of activity were made at each of three different count levels. Three images of the Amersham brain phantom were obtained at each of three count levels, roughly corresponding to the simulation levels. Image reconstruction involved a modified Shepp-Logan filter with and without attenuation correction. The scaling constant in the Budinger equation was shown to vary little over the count range used with a mean value of 23 for uncorrected phantom data and 27 for corrected phantom data, corresponding to RMS noise levels of 7%-15%. The variance due to noise was calculated as a percentage of the variance obtained for 53 normal control studies following image registration and normalization. Values of 54% for uncorrected images and 67% for corrected images were obtained. For 10 normal controls a repeated study was performed. The ratio of within-subject to (single sample) between-subject variance was determined as 73% for uncorrected images and 78% for corrected images.     

Detection of Clostridium difficile toxin: comparison of enzyme immunoassay results with results obtained by cytotoxicity assay

Several kinds of laboratory techniques are available to detect Clostridium difficile toxin in fecal samples. Because questions have been raised about the reliability of immunoassays compared to the accepted standard, cytotoxicity assay, we studied three enzyme immunoassays (EIAs) and one rapid EIA, which demonstrated relatively good sensitivities and specificities compared to cytotoxicity assay.     

Physical activity monitoring based on accelerometry: validation and comparison with video observation

The objective of this feasibility study is to evaluate the use of the ‘Physilog’ device, an ambulatory physical-activity recorder based on acceleration measurement, for the monitoring of daily physical activities. Accelerations measured at the level of the chest and the thigh are recorded by Physilog over a period of 1 h in five normal subjects. A specially designed studio-like room allowing the performance of most usual activities of everyday life is used. A video film synchronised with the Physilog is obtained for each subject to check the accuracy of the data derived from Physilog. Based on the analysis of the average and the deviation of the acceleration signal, an algorithm is developed to classify the activities in four categories, i.e. lying, sitting, standing and locomotion. Compared with the video observations, the results from the algorithm show an overall misclassification of 10.7%, which is mainly due to confusion between dynamic activities and the standing posture. In contrast, the misclassification between postures is negligible. It is concluded that Physilog can be used in the clinical setting for the reliable measurement and long-term recording of most-usual physical activities.     

Physical activity monitoring based on accelerometry: validation and comparison with video observation

The objective of this feasibility study is to evaluate the use of the 'Physilog' device, an ambulatory physical-activity recorder based on acceleration measurement, for the monitoring of daily physical activities. Accelerations measured at the level of the chest and the thigh are recorded by Physilog over a period of 1 h in five normal subjects. A specially designed studio-like room allowing the performance of most usual activities of everyday life is used. A video film synchronised with the Physilog is obtained for each subject to check the accuracy of the data derived from Physilog. Based on the analysis on the average and the deviation of the acceleration signal, an algorithm is developed to classify the activities in four categories, i.e. lying, sitting, standing and locomotion. Compared with the video observations, the results from the algorithm show an overall misclassification of 10.7%, which is mainly due to confusion between dynamic activities and the standing posture. In contrast, the misclassification between postures is negligible. It is concluded that Physilog can be used in the clinical setting for the reliable measurement and long-term recording of most usual physical activities.     

A comparison of cervical scrapes for HPV typing by dot-blot hybridization obtained by wood and plastic spatulas

A cross-over study was designed to determine whether the type of spatula used to collect cervical cells influences the ability of dot-blot hybridization to detect HPV DNA. Fifty-nine patients had a cervical scrape with a wood spatula first and a plastic spatula second: 60 were scraped in the inverse order. The order of sampling did not affect the HPV DNA positivity rate, which was nearly similar for both wood and plastic spatulas (30 and 32%, respectively). Wood spatulas collected more cells and greater than 1 x 10(5) cells more often than plastic spatulas (P = 0.001 and 0.06, respectively). Non-purple (negative) dots were more frequent in samples obtained by wood than by plastic spatulas (P = 0.001). The study showed that cervical cell collection by wood spatulas is preferred as they harvest more cells, thus optimizing the sensitivity of the hybridization method, and the spatulas are also more economical. Although they yielded more non-purple dots, a reduction in these dots by using plastic spatulas did not result in a significantly increased HPV positivity rate.     

Benzene increases aneuploidy in the lymphocytes of exposed workers: a comparison of data obtained by fluorescence in situ hybridization in interphase and metaphase cells

Benzene is an established human leukemogen that increases the level of chromosome aberrations in lymphocytes of exposed workers. Numerical aberrations (aneusomy) can be observed by fluorescence in situ hybridization (FISH) in both interphase and metaphase cells. Whereas interphase FISH allows nondividing cells to be analyzed, one advantage of metaphase FISH is that it can also detect structural changes. The present study compares the abilities of metaphase and interphase FISH to detect aneusomy of chromosomes 7 and 8 in healthy benzene-exposed human subjects. Metaphase and interphase cells from the peripheral blood of 43 workers exposed to benzene (median = 31 ppm, 8-hr TWA) and 44 frequency-matched controls were analyzed by FISH. Normal diploid cells contained two hybridization signals, whereas those that were potentially monosomic contained one, trisomic 3 and tetrasomic 4. The frequency of cells with one hybridization signal for chromosome 7 in metaphase spreads rose from 2.72 +/- 0.19 (%, mean +/- SE) in controls to 3.79 +/- 0.63 in workers exposed to 31 or fewer ppm benzene and 5.9 +/- 0.85 in those exposed to more than 31 ppm (P(trend) < 0.0001). No similar dose-dependent increase in the frequency of cells with one hybridization signal was observed by interphase FISH, probably because of probe overlap artifact. Although significant dose-dependent increases in the frequency of cells with three hybridization signals for chromosome 7 were detected by both methods in the higher-exposed group, a larger, more significant difference was detected by metaphase FISH between controls and workers exposed to 31 or fewer ppm. Similar data were obtained for chromosome 8. Interphase and metaphase FISH were moderately correlated for three hybridization signals but not for one hybridization signal in chromosome 7 or 8. In general, metaphase FISH was more sensitive in detecting both monosomy and trisomy in the lymphocytes of exposed workers.     

Fine-needle biopsy: comparison of cellular yield with and without aspiration

Fine-needle biopsies with and without aspiration were performed on 103 lesions, and the cellular yields obtained by the two techniques were compared. A scoring system was devised by which the amount of material obtained in each needle stick was estimated. The mean score/stick for fine-needle biopsy with aspiration was 2.7; for biopsy without aspiration, it was 2.9. The results of this study indicate that there is no significant difference in the amount of cellular material obtained by the two techniques. It is concluded that needle biopsy without aspiration may be performed routinely with good results.     

Novel findings with reassessment of exome data: implications for validation testing and interpretation of genomic data

PurposeThe objective of this study was to assess the ability of our laboratory’s exome-sequencing test to detect known and novel sequence variants and identify the critical factors influencing the interpretation of a clinical exome test.MethodsWe developed a two-tiered validation strategy: (i) a method-based approach that assessed the ability of our exome test to detect known variants using a reference HapMap sample, and (ii) an interpretation-based approach that assessed our relative ability to identify and interpret disease-causing variants, by analyzing and comparing the results of 19 randomly selected patients previously tested by external laboratories.ResultsWe demonstrate that this approach is reproducible with >99% analytical sensitivity and specificity for single-nucleotide variants and indels <10 bp. Our findings were concordant with the reference laboratories in 84% of cases. A new molecular diagnosis was applied to three cases, including discovery of two novel candidate genes.ConclusionWe provide an assessment of critical areas that influence interpretation of an exome test, including comprehensive phenotype capture, assessment of clinical overlap, availability of parental data, and the addressing of limitations in database updates. These results can be used to inform improvements in phenotype-driven interpretation of medical exomes in clinical and research settings.