Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome in which the known susceptibility genes (DKC1, TERC, and TERT) belong to the telomere maintenance pathway; patients with DC have very short telomeres. We used multicolor flow fluorescence in situ hybridization analysis of median telomere length in total blood leukocytes, granulocytes, lymphocytes, and several lymphocyte subsets to confirm the diagnosis of DC, distinguish patients with DC from unaffected family members, identify clinically silent DC carriers, and discriminate between patients with DC and those with other bone marrow failure disorders. We defined "very short" telomeres as below the first percentile measured among 400 healthy control subjects over the entire age range. Diagnostic sensitivity and specificity of very short telomeres for DC were more than 90% for total lymphocytes, CD45RA+/CD20- naive T cells, and CD20+ B cells. Granulocyte and total leukocyte assays were not specific; CD45RA- memory T cells and CD57+ NK/NKT were not sensitive. We observed very short telomeres in a clinically normal family member who subsequently developed DC. We propose adding leukocyte subset flow fluorescence in situ hybridization telomere length measurement to the evaluation of patients and families suspected to have DC, because the correct diagnosis will substantially affect patient management.

Clinical performance of a new laser fluorescence device for detection of occlusal caries lesions in permanent molars

OBJECTIVES: To determine the clinical performance of a laser fluorescence device (DIAGNOdent pen, KaVo) to discriminate between different occlusal caries depths (D(0)-D(1-4); D(0-2)-D(3,4)) in permanent molars. METHODS: In this prospective, randomized two-centre-study 120 sound/uncavitated carious sites in 120 patients were measured after visual and radiographic caries assessment. In cases of operative intervention (n=86), the lesion depths after caries removal were recorded (reference). In cases of preventive intervention (n=34), the sites were reassessed visually/radiographically after 12 months to verify the status assessed before (reference). The discrimination performance was determined statistically (Mann-Whitney test, Spearman's rho coefficient, and areas under the receiver operating characteristic curves (AUCs)). Sensitivities (SE) and specificities (SP) were plotted as a function of the measured values and cut-off values for the mentioned thresholds suggested. RESULTS: Sound sites (n=13) had significantly minor fluorescence values than carious sites (n=107) (P<0.0001) as had sites with no/enamel caries (n=63) compared to dentinal caries (n=57). The AUCs for the same discriminations were 0.92 and 0.78 (P<0.001). For the D(0)-D(1-4) threshold, a cut-off at a value of 12 (SE: 0.88, SP: 0.85) and for the D(0-2)-D(3,4) threshold at 25 (SE: 0.67, SP: 0.79) can be suggested. A moderate positive correlation between the measurements and the caries depths was calculated (rho=+0.57, P=0.01). CONCLUSION: Within this study, the device's discrimination performance for different caries depths was moderate to very good and it may be recommended as adjunct tool in the diagnosis of occlusal caries.

The influence of zero-value subtraction on the performance of two laser fluorescence devices for detecting occlusal caries in vitro

BACKGROUND: The aim of this study was to evaluate the influence of zero-value subtraction on the performance of two laser fluorescence (LF) devices developed to detect occlusal caries. METHODS: The authors selected 119 permanent molars. Two examiners assessed three areas (cuspal, middle and cervical) of both mesial and distal portions of the buccal surface and one occlusal site using an LF device and an LF pen. For each tooth, the authors subtracted the value measured in the cuspal, middle and cervical areas in the buccal surface from the value measured in the respective occlusal site. RESULTS: The authors observed differences among the readings for both devices in the cuspal, middle and cervical areas in the buccal surface as well as differences for both devices with and without the zero-value subtraction in the occlusal surface. When the authors did not perform the zero-value subtraction, they found statistically significant differences for sensitivity and accuracy for the LF device. When this was done with the LF pen, specificity increased and sensitivity decreased significantly. CONCLUSIONS: For the LF device, the zero-value subtraction decreased the sensitivity. For this reason, the authors concluded that clinicians can obtain measures with the LF device effectively without using zero-value subtraction. For the LF pen, however, the absence of the zero-value subtraction changed both the sensitivity and specificity, and so the authors concluded that clinicians should not eliminate this step from the procedure. CLINICAL IMPLICATIONS: When using the LF device, clinicians might not need to perform the zero-value subtraction; however, for the LF pen, clinicians should do so.

The influence of zero value subtraction on the performance of a new laser fluorescence device for approximal caries detection

The aim of this study was to assess the influence of the zero value subtraction on the performance of laser fluorescence (LFpen) for approximal caries detection. Three areas (cuspal, middle and cervical) of both mesial and distal buccal surfaces of 78 permanent molars were assessed using both wedge-shaped (WDG) and tapered wedge-shaped (TWDG) tips. With the addition of the average, one cut-off value for each area was obtained and the performance was assessed. The areas under the receiver operating characteristics (ROC) curve, specificity, sensitivity and accuracy with and without the zero value subtraction were calculated. The McNemar test revealed a statistically significant difference for specificity at thresholds D(1), D(2) and D(3) (WDG) and D(1) and D(2) (TWDG) when the zero value subtraction was not performed. Influence of the zero value subtraction on the LFpen performance was observed for approximal caries detection. However, when modified cut-off values were used, the zero value subtraction could be eliminated.

The influence of pit and fissure sealants on infrared fluorescence measurements

The aim of this in vitro study was to evaluate the influence of pit and fissure sealants on fluorescence readings using lasers. We selected 166 permanent molars and randomly divided them into 4 groups which were each treated with a different sealant (a commercially available clear sealant, 2 opaque sealants and an experimental nanofilled clear sealant). The teeth were independently measured twice by 2 experienced dentists using conventional laser fluorescence (LF) and a laser fluorescence pen device (LFpen), before and after sealing, and again after thermocycling to simulate the thermal stressing between the tooth and the dental materials. Friedman test showed no statistically significant changes using LF and LFpen for the commercial clear sealant group, although values tended to increase after sealing. However, the values increased significantly after thermocycling. There was a statistically significant decrease in fluorescence after application of opaque sealants. After application of the experimental nanofilled clear sealant, LF values increased only after thermocycling, whereas the LFpen values increased after sealing and after thermocycling as well. The intraclass correlation coefficient ranged from 0.87 to 0.96 for interexaminer and 0.82 to 0.94 for intraexaminer reproducibility. It was shown that pit and fissure sealants influence LF and LFpen readings, with the values increasing or decreasing according to the material used. In conclusion, both laser fluorescence devices could be useful as an adjunct to detect occlusal caries under unfilled clear sealants. Nevertheless, surfaces sealed with clear nanofilled material could be assessed using only the LF device.

Performance of fluorescence methods, radiographic examination and ICDAS II on occlusal surfaces in vitro

This study compared the performance of fluorescence-based methods, radiographic examination, and International Caries Detection and Assessment System (ICDAS) II on occlusal surfaces. One hundred and nineteen permanent human molars were assessed twice by 2 experienced dentists using the laser fluorescence (LF and LFpen) and fluorescence camera (FC) devices, ICDAS II and bitewing radiographs (BW). After measuring, the teeth were histologically prepared and assessed for caries extension. The sensitivities for dentine caries detection were 0.86 (FC), 0.78 (LFpen), 0.73 (ICDAS II), 0.51 (LF) and 0.34 (BW). The specificities were 0.97 (BW), 0.89 (LF), 0.65 (ICDAS II), 0.63 (FC) and 0.56 (LFpen). BW presented the highest values of likelihood ratio (LR)+ (12.47) and LR- (0.68). Rank correlations with histology were 0.53 (LF), 0.52 (LFpen), 0.41 (FC), 0.59 (ICDAS II) and 0.57 (BW). The area under the ROC curve varied from 0.72 to 0.83. Inter- and intraexaminer intraclass correlation values were respectively 0.90 and 0.85 (LF), 0.93 and 0.87 (LFpen) and 0.85 and 0.76 (FC). The ICDAS II kappa values were 0.51 (interexaminer) and 0.61 (intraexaminer). The BW kappa values were 0.50 (interexaminer) and 0.62 (intraexaminer). The Bland and Altman limits of agreement were 46.0 and 38.2 (LF), 55.6 and 40.0 (LFpen) and 1.12 and 0.80 (FC), for intra- and interexaminer reproducibilities. The posttest probability for dentine caries detection was high for BW and LF. In conclusion, LFpen, FC and ICDAS II presented better sensitivity and LF and BW better specificity. ICDAS II combined with BW showed the best performance and is the best combination for detecting caries on occlusal surfaces.

Study of and design procedure for dual circularly polarized waveguide slot arrays

For the past sixty years, waveguide slot radiator arrays have played a critical role in microwave radar and communication systems. They feature a well-characterized antenna element capable of direct integration into a low-loss feed structure with highly developed and inexpensive manufacturing processes. Waveguide slot radiators comprise some of the highest performance—in terms of side-lobe-level, efficiency, etc. — antenna arrays ever constructed. A wealth of information is available in the open literature regarding design procedures for linearly polarized waveguide slots. By contrast, despite their presence in some of the earliest published reports, little has been presented to date on array designs for circularly polarized (CP) waveguide slots. Moreover, that which has been presented features a classic traveling wave, efficiency-reducing beam tilt. This work proposes a unique CP waveguide slot architecture which mitigates these problems and a thorough design procedure employing widely available, modern computational tools. The proposed array topology features simultaneous dual-CP operation with grating-lobe-free, broadside radiation, high aperture efficiency, and good return loss. A traditional X-Slot CP element is employed with the inclusion of a slow wave structure passive phase shifter to ensure broadside radiation without the need for performance-limiting dielectric loading. It is anticipated this technology will be advantageous for upcoming polarimetric radar and Ka-band SatCom systems. The presented design methodology represents a philosophical shift away from traditional waveguide slot radiator design practices. Rather than providing design curves and/or analytical expressions for equivalent circuit models, simple first-order design rules – generated via parametric studies — are presented with the understanding that device optimization and design will be carried out computationally. A unit-cell, S-parameter based approach provides a sufficient reduction of complexity to permit efficient, accurate device design with attention to realistic, application-specific mechanical tolerances. A transparent, start-to-finish example of the design procedure for a linear sub-array at X-Band is presented. Both unit cell and array performance is calculated via finite element method simulations. Results are confirmed via good agreement with finite difference, time domain calculations. Array performance exhibiting grating-lobe-free, broadside-scanned, dual-CP radiation with better than 20 dB return loss and over 75% aperture efficiency is presented.

Non-invasive method to predict viscosity and size using total internal reflection fluorescence microscopy (TIRFM) system

This study develops an automated analysis tool by combining total internal reflection fluorescence microscopy (TIRFM), an evanescent wave microscopic imaging technique to capture time-sequential images and the corresponding image processing Matlab code to identify movements of single individual particles. The developed code will enable us to examine two dimensional hindered tangential Brownian motion of nanoparticles with a sub-pixel resolution (nanoscale). The measured mean square displacements of nanoparticles are compared with theoretical predictions to estimate particle diameters and fluid viscosity using a nonlinear regression technique. These estimated values will be confirmed by the diameters and viscosities given by manufacturers to validate this analysis tool. Nano-particles used in these experiments are yellow-green polystyrene fluorescent nanospheres (200 nm, 500 nm and 1000 nm in diameter (nominal); 505 nm excitation and 515 nm emission wavelengths). Solutions used in this experiment are de-ionized (DI) water, 10% d-glucose and 10% glycerol. Mean square displacements obtained near the surface shows significant deviation from theoretical predictions which are attributed to DLVO forces in the region but it conforms to theoretical predictions after ~125 nm onwards. The proposed automation analysis tool will be powerfully employed in the bio-application fields needed for examination of single protein (DNA and/or vesicle) tracking, drug delivery, and cyto-toxicity unlike the traditional measurement techniques that require fixing the cells. Furthermore, this tool can be also usefully applied for the microfluidic areas of non-invasive thermometry, particle tracking velocimetry (PTV), and non-invasive viscometry.

The influence of PVC wrapping on the performance of two laser fluorescence devices on occlusal surfaces in vitro

OBJECTIVE: The aim of this study was to determine the influence of polyvinyl chloride (PVC) wrapping on the performance of two laser fluorescence devices (LF and LFpen) by assessing tooth occlusal surfaces. BACKGROUND DATA: Protection of their tips may influence LF measurements. To date there are no studies evaluating the influence of this protection on the performance of the LFpen on permanent teeth, or comparing it to the original LF device. MATERIALS AND METHODS: One hundred nineteen permanent molars were assessed by two experienced dentists using the LF and the LFpen devices, both with and without PVC wrapping. The teeth were histologically prepared and assessed for caries extension. RESULTS: The LF values with and without PVC wrapping were significantly different. For both LF devices, the sensitivity and accuracy were lower when the PVC wrapping was used. The specificity was statistically significantly higher for the LFpen with PVC. No difference was found between the areas under the ROC curves with and without PVC wrapping. The ICC showed excellent interexaminer agreement. The Bland and Altman method showed a range between the upper and the lower limits of agreement of 63.4 and 57.8 units for the LF device, and 49.4 and 74.2 for the LFpen device, with and without PVC wrapping, respectively. CONCLUSIONS: We found an influence of the PVC wrapping on the performance of the LF and LFpen devices. However, since its influence on detection of occlusal caries lesions is considered for, the use of one PVC layer is suggested to avoid cross-contamination in clinical practice.

In vitro comparison of laser fluorescence performance with visual examination for detection of occlusal caries in permanent and primary molars

The aim of this study was to compare the performance of the DIAGNOdent 2095 with visual examination for occlusal caries detection in permanent and primary molars. The sample comprised 148 permanent human molars and 179 primary human molars. The samples were measured and visually examined three times by two examiners. After measurement, the teeth were histologically prepared and assessed for caries extension. Sensitivity, specificity, accuracy and area under the receiver operating characteristics (ROC) curve were calculated. Intra-class correlation (ICC), unweighted kappa and the Bland and Altman method were used to assess inter- and intra-examiner reproducibility. DIAGNOdent showed higher specificity and lower sensitivity than did visual examination. The ICC values indicated an excellent agreement between the examinations. Kappa values varied from good to excellent for DIAGNOdent but from poor to good for visual examination. In conclusion, the DIAGNOdent may be a useful adjunct to conventional methods for occlusal caries detection.

In vivo evaluation of laser fluorescence performance using different cut-off limits for occlusal caries detection

The aim of this in vivo study was to evaluate the performance of laser fluorescence (LF) comparing different cut-off limits for occlusal caries detection. One hundred and thirty first permanent molars were selected. Visual examination and LF assessments were performed independently. The extent of caries was assessed after operative intervention. New cut-off limits were established and compared with those proposed by the manufacturer and by Lussi et al. (Eur J Oral Sci 109:14-19, 2001). Similar sensitivity and higher specificity was found at D(2) (considering as disease only dentin caries) when the LF cut-off limits proposed by Lussi et al. and the new one were compared. At the D(3) threshold (considering as disease only deep dentin caries), no statistically significant difference among the cut-off limits for sensitivity was found. However, the new cut-off limits showed higher specificity. The LF device provided good ability to detect dentin caries lesions. Furthermore, the new cut-off limits and the values proposed by Lussi et al. could be suggested for the in vivo detection of occlusal caries.