Enhanced Real-Time Pose Estimation for Closed-Loop Robotic Manipulation of Magnetically Actuated Capsule Endoscopes.

Pose estimation methods for robotically guided magnetic actuation of capsule endoscopes have recently enabled trajectory following and automation of repetitive endoscopic maneuvers. However, these methods face significant challenges in their path to clinical adoption including the presence of regions of magnetic field singularity, where the accuracy of the system degrades, and the need for accurate initialization of the capsule's pose. In particular, the singularity problem exists for any pose estimation method that utilizes a single source of magnetic field if the method does not rely on the motion of the magnet to obtain multiple measurements from different vantage points. We analyze the workspace of such pose estimation methods with the use of the point-dipole magnetic field model and show that singular regions exist in areas where the capsule is nominally located during magnetic actuation. Since the dipole model can approximate most magnetic field sources, the problem discussed herein pertains to a wider set of pose estimation techniques. We then propose a novel hybrid approach employing static and time-varying magnetic field sources and show that this system has no regions of singularity. The proposed system was experimentally validated for accuracy, workspace size, update rate and performance in regions of magnetic singularity. The system performed as well or better than prior pose estimation methods without requiring accurate initialization and was robust to magnetic singularity. Experimental demonstration of closed-loop control of a tethered magnetic device utilizing the developed pose estimation technique is provided to ascertain its suitability for robotically guided capsule endoscopy. Hence, advances in closed-loop control and intelligent automation of magnetically actuated capsule endoscopes can be further pursued toward clinical realization by employing this pose estimation system.

Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials.

BACKGROUND: Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62-164 bp). RESULTS: Results from the Bioanalyzer and TaqMan data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70 degrees C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification. CONCLUSION: Modification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts.

Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization.

Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.