COMP Report: CPQR technical quality control guidelines for use of positron emission tomography/computed tomography in radiation treatment planning.

Positron emission tomography with x-ray computed tomography (PET/CT) is increasingly being utilized for radiation treatment planning (RTP). Accurate delivery of RT therefore depends on quality PET/CT data. This study covers quality control (QC) procedures required for PET/CT for diagnostic imaging and incremental QC required for RTP. Based on a review of the literature, it compiles a list of recommended tests, performance frequencies, and tolerances, as well as references to documents detailing how to perform each test. The report was commissioned by the Canadian Organization of Medical Physicists as part of the Canadian Partnership for Quality Radiotherapy initiative.

The Operation of Canada's Only Virtually Operated Radiation Oncology Service During the COVID-19 Pandemic.

PURPOSE: Our institution operates a remote radiation oncology service in Northern Ontario, Canada. Since the start of the coronavirus disease 2019 pandemic, this center has operated without radiation oncologists on site owing to safety precautions, and this study seeks to understand the effect of this shift. METHODS AND MATERIALS: Departmental level data reports were used to investigate differences in metrics between April to May of 2019 and April to May 2020. These metrics include the total number of referrals received, average wait time from referral to consult, the number of cases that underwent peer review before beginning treatment, the total number of fractions given over each period, patient-reported outcomes, and patient satisfaction. We also examined the importance of physical examinations and the use of SABR treatment. RESULTS: There was an observed decrease in the number of referrals received, total number of fractions administered, and number of patients providing patient-reported outcomes. We observed no change in patient wait times, cases undergoing peer review before commencing treatment, or overall patient satisfaction. Challenges were identified in the collection of patient- reported outcomes and the conduction of physical examinations. CONCLUSIONS: This paper provides proof of concept that a radiation clinic can function entirely virtually in the short term without sacrificing patient satisfaction, efficiency, or safety.

Understanding the psychological and social influences on office workers taking breaks; a thematic analysis.

Objective: There is a growing trend whereby office workers refrain from taking breaks at work. Previous research has not explored how employees understand the enablers and barriers to taking breaks. This study explored how office-based workers describe their behaviour in relation to, and perceive the notion of, taking breaks.Design: Five focus groups were held with 27 employees of differing levels of seniority at a local authority in the UK. Inductive thematic analysis was employed with the researchers maintaining a deliberate stance of curiosity towards the data, allowing for reflexivity and awareness of preconceptions towards the research.Results: The analysis identified 5 key themes: the non-binary nature of taking breaks at work, the influence of social and work relationships, the superordination of work over breaks and health, contradictory feelings of guilt and anxiety and being 'fair game' for work related matters if you remain at your desk at break times.Conclusion: This paper suggests that the complex relationships that people have with taking breaks, with others and with their physical environment should be taken into account when trying to understand break-taking behaviour. Based on these findings, suggestions for further research and potential health-related policy and organisational changes are made.

Effective Partnership in Community-Based Health Promotion: Lessons from the Health Literacy Partnership.

This paper aims to explore key elements needed to successfully develop healthy partnerships and collaborative working in community-based health promotion. It draws upon the lessons learned from a case study with the Health Literacy Partnership in Stoke-on-Trent, UK in developing the health literacy strategy in the area. The process was underpinned by respect for diverse yet complementary perspectives and skills from the grassroots up. This involved engagement with key stakeholders, development and support for community projects, and sharing of good practice with other national and local organizations. Stakeholders involved in developing the strategy also had a keen interest in health literacy and a strong commitment to promoting health and well-being in the area. Through patience, perseverance, and continuous open communication and learning, the health literacy strategy in Stoke-on-Trent, UK is beginning to have a ripple effect into local practice, and will potentially influence policy in the future.

Trying to fall asleep while catastrophising: what sleep-disordered adolescents think and feel.

OBJECTIVE: Catastrophising is a repetitive cognitive process related to sleep disturbance in adult insomnia patients. More recently catastrophising has been associated with increased sleep disturbances in community samples of children and adolescents, with this association mediated by anxiety and depression. However, there currently is no evidence of these processes outside of community samples; impeding our ability to draw clinical conclusions. Knowledge on such dysfunctional cognitive processes in adolescents experiencing sleep disturbance would be clinically beneficial in aetiology and intervention. Our research examined the link between catastrophising, anxiety, depression and sleep latency in a sample of sleep-disordered adolescents. We also explored specific catastrophising themes which may impact the sleep latency of these adolescents. METHOD: Forty adolescents (age=15.1±1.5 years, 53% boys) diagnosed with delayed sleep phase disorder completed a 7-day sleep diary, along with measures of anxiety and depression and a catastrophising interview with a trained sleep therapist. RESULTS: Several catastrophisation themes were generated, the most common concerning interpersonal and performance aspects of school. Bootstrapping analyses showed depression did not mediate the relationship between catastrophising and sleep; however, an indirect relationship was found between catastrophising, anticipatory anxiety, and sleep latency. CONCLUSION: These findings have implications for the role of dysfunctional thinking in prolonging sleep onset for adolescents and providing a clinical framework for health professionals when assessing and treating adolescents with delayed sleep timing.

Interview: challenges faced by the modern bioanalytical laboratory.

Timothy Sangster (Charles River Laboratories) and Mike Oliver (Thermo Fisher Scientific) speak to Thomas Payne at Bioanalysis in September 2012 about the challenges faced by the modern bioanalytical laboratory. Timothy Sangster has been with Charles River Laboratories since September 2009. Having worked for Quintiles, Pharmacia, Astrazeneca and Huntingdon Life Sciences, he has gained experience over 17 years in both CRO and pharmaceutical environments, and also in both Europe and the USA. Specific areas of interest over the past years have been microchromatography, sample preparation and matrix effects, to name a few. Mike Oliver has held the position of Global Product Manager for sample preparation at Thermo Fisher Scientific since 2010, being responsible for the development and introduction of new innovative technologies such as SOLA™ to the market place. Prior to this role, Mike has worked for two leading MS vendors over a 9­year period, being responsible for biotechnology sales within the UK and providing application solutions for proteomic and metabolic workflows based on high-resolution LC-MS platforms, respectively. Mike holds a PhD in MS and Biochemistry from the MS Research Unit, University of Wales, Swansea, UK.

Donating in good faith or getting into trouble Religion and organ donation revisited.

There is worldwide shortage of organs for solid-organ transplantation. Many obstacles to deceased and live donation have been described and addressed, such as lack of understanding of the medical process, the issue of the definition of brain death, public awareness of the need for transplants, and many others. However, it is clear that the striking differences in deceased and live donation rates between different countries are only partly explained by these factors and many cultural and social reasons have been invoked to explain these observations. We believe that one obstacle to both deceased and live donation that is less well appreciated is that of religious concerns. Looking at the major faiths and religions worldwide, it is reassuring to see that most of them encourage donation. However, there is also scepticism amongst some of them, often relating to the concept of brain death and/or the processes surrounding death itself. It is worthwhile for transplant teams to be broadly aware of the issues and also to be mindful of resources for counselling. We believe that increased awareness of these issues within the transplant community will enable us to discuss these openly with patients, if they so wish.

Clinical significance of multi-leaf collimator positional errors for volumetric modulated arc therapy.

BACKGROUND AND PURPOSE: Multi-leaf collimator (MLC) positional errors occur during intensity modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT) deliveries. The impact of such errors has been evaluated for IMRT but not VMAT. The purpose of this work is to understand how random and systematic VMAT MLC positional errors affect the patient dose distribution. MATERIALS AND METHODS: Eight head and neck single arc (360°) VMAT treatment plans were created. Random and two types of systematic MLC errors were simulated for error magnitudes of 0.25, 0.5, 1, 2 and 5mm. The two types of systematic MLC errors were: (1) MLC banks are shifted in the same direction (left or right) and (2) MLC banks are shifted in opposing directions resulting in smaller or larger field shapes. The MLC errors were simulated, for all control points, on both banks of active MLC leaves only. RESULTS: There is a linear correlation of MLC errors with gEUD for all error types. The gEUD dose sensitivities with MLC error for the PTV70 were -0.2, -0.9, -2.8 and 1.9 Gy/mm for random, systematic shift, systematic close and systematic open MLC errors, respectively. The sensitivity of VMAT plans to MLC positional errors was similar to those of IMRT plans with less than 50 segments but much less than those created for a step and shoot with more than 50 segments or sliding-window delivery technique. To maintain the PTV70 to within 2% would require that MLC open/close errors be within 0.6mm. CONCLUSIONS: Radiation therapy centers should have adequate quality assurance programs in place to assess open/close MLC errors (i.e. leaf gap errors) as they tend to be more impactful than random or systematic MLC shift errors.

Comparing planning time, delivery time and plan quality for IMRT, RapidArc and Tomotherapy.

The purpose of this study is to examine plan quality, treatment planning time, and estimated treatment delivery time for 5- and 9-field sliding window IMRT, single and dual arc RapidArc, and tomotherapy. For four phantoms, 5- and 9-field IMRT, single and dual arc RapidArc and tomotherapy plans were created. Plans were evaluated based on the ability to meet dose-volume constraints, dose homogeneity index, radiation conformity index, planning time, estimated delivery time, integral dose, and volume receiving more than 2 and 5 Gy. For all of the phantoms, tomotherapy was able to meet the most optimization criteria during planning (50% for P1, 67% for P2, 0% for P3, and 50% for P4). RapidArc met less of the optimization criteria (25% for P1, 17% for P2, 0% for P3, and 0% for P4), while IMRT was never able to meet any of the constraints. In addition, tomotherapy plans were able to produce the most homogeneous dose. Tomotherapy plans had longer planning time, longer estimated treatment times, lower conformity index, and higher integral dose. Tomotherapy plans can produce plans of higher quality and have the capability to conform dose distributions better than IMRT or RapidArc in the axial plane, but exhibit increased dose superior and inferior to the target volume. RapidArc, however, is capable of producing better plans than IMRT for the test cases examined in this study.

Evaluation of optimization strategies and the effect of initial conditions on IMAT optimization using a leaf position optimization algorithm.

Intensity-modulated arc therapy (IMAT) is a rotational variant of intensity-modulated radiation therapy (IMRT) that can be implemented with or without angular dose rate variation. The purpose of this study is to assess optimization strategies and initial conditions using a leaf position optimization (LPO) algorithm altered for variable dose rate IMAT. A concave planning target volume (PTV) with a central cylindrical organ at risk (OAR) was used in this study. The initial IMAT arcs were approximated by multiple static beams at 5 degrees angular increments where multi-leaf collimator (MLC) leaf positions were determined from the beam's eye view to irradiate the PTV but avoid the OAR. For the optimization strategy, two arcs with arc ranges of 280 degrees and 150 degrees were employed and plans were created using LPO alone, variable dose rate optimization (VDRO) alone, simultaneous LPO and VDRO and sequential combinations of these strategies. To assess the MLC initialization effect, three single 360 degrees arc plans with different initial MLC configurations were generated using the simultaneous LPO and VDRO. The effect of changing optimization degrees of freedom was investigated by employing 3 degrees , 5 degrees and 10 degrees angular sampling intervals for the two 280 degrees , two 150 degrees and single arc plans using LPO and VDRO. The objective function value, a conformity index, a dose homogeneity index, mean dose to OAR and normal tissues were computed and used to evaluate the treatment plans. This study shows that the best optimization strategy for a concave target is to use simultaneous MLC LPO and VDRO. We found that the optimization result is sensitive to the choice of initial MLC aperture shapes suggesting that an LPO-based IMAT plan may not be able to overcome local minima for this geometry. In conclusion, simultaneous MLC leaf position and VDRO are needed with the most appropriate initial conditions (MLC positions, arc ranges and number of arcs) for IMAT.

Experimental measurements and Monte Carlo simulations for dosimetric evaluations of intrafraction motion for gated and ungated intensity modulated arc therapy deliveries.

Respiratory gated radiation therapy allows for a smaller margin expansion for the planning target volume (PTV) to account for respiratory induced motion and is emerging as a common method to treat lung and liver tumors. We investigated the dosimetric effect of free motion and gated delivery for intensity modulated arc therapy (IMAT) with experimental measurements and Monte Carlo simulations. The impact of PTV margin and duty cycle for gated delivery is studied with Monte Carlo simulations. A motion phantom is used for this study. Two sets of contours were drawn on the mid-inspiration CT scan of this motion phantom. For each set of contours, an IMAT plan to be delivered with constant dose rate was created. The plans were generated on a CT scan of the phantom in the static condition with 3 mm PTV margin and applied to the motion phantom under four conditions: static, full superior-inferior (SI) motion (A = 1 cm, T = 4 s) and gating conditions (25% and 50% duty cycles) with full SI motion. A 6 by 15 cm piece of radiographic film was placed in the sagittal plane of the phantom and then irradiated under all measurement conditions. Film calibration was performed with a step-wedge method to convert optical density to dose. Gated IMAT delivery was first validated in 2D by comparing static film with that from gating and full motion. A previously verified simulation tool for IMRT that takes the log files from the multileaf collimator (MLC) controller and the gating system were adapted to simulate the delivered IMAT treatment for full 3D dosimetric analysis. The IMAT simulations were validated against the 2D film measurements. The resultant IMAT simulations were evaluated with dose criteria, dose-volume histograms and 3D gamma analysis. We validated gated IMAT deliveries when we compared the static film with the one from gating using 25% duty cycle using 2D gamma analysis. Within experimental and setup uncertainties, film measurements agreed with their corresponding simulated plans using 2D gamma analysis. Finally, when planning with margins designed for gating with 25% duty cycle and applying 50% or no gating during treatment, the dose differences in D(min,) D(99%) and D(95%) of the clinical target volume can be up to 27 cGy, 20 cGy and 18 cGy, respectively, for a plan with 200 cGy prescription dose. We have experimentally delivered gated IMAT with constant dose rate to a motion phantom and assessed their accuracies with film dosimetry and Monte Carlo simulations. Film dosimetry demonstrated that 25% gating and static plans are within 2%, 2 mm. The Monte Carlo simulation method was employed to generate dose delivered in 3D to a motion phantom, and the dosimetric results were reported. Since our film measurements agreed well with Monte Carlo simulations, we can reliably use this simulation tool to further study the dosimetric effects of target motion and effectiveness of gating for IMAT deliveries.

Incorporating geometric ray tracing to generate initial conditions for intensity modulated arc therapy optimization.

PURPOSE AND BACKGROUND: Intensity modulated arc therapy (IMAT) is a rotational variant of Intensity modulated radiation therapy (IMRT) that is achieved by allowing the multileaf collimator (MLC) positions to vary as the gantry rotates around the patient. This work describes a method to generate an IMAT plan through the use of a fast ray tracing technique based on dosimetric and geometric information for setting initial MLC leaf positions prior to final IMAT optimization. METHODS AND MATERIALS: Three steps were used to generate an IMAT plan. The first step was to generate arcs based on anatomical contours. The second step was to generate ray importance factor (RIF) maps by ray tracing the dose distribution inside the planning target volume (PTV) to modify the MLC leaf positions of the anatomical arcs to reduce the maximum dose inside the PTV. The RIF maps were also segmented to create a new set of arcs to improve the dose to low dose voxels within the PTV. In the third step, the MLC leaf positions from all arcs were put through a leaf position optimization (LPO) algorithm and brought into a fast Monte Carlo dose calculation engine for a final dose calculation. The method was applied to two phantom cases, a clinical prostate case and the Radiological Physics Center (RPC)'s head and neck phantom. The authors assessed the plan improvements achieved by each step and compared plans with and without using RIF. They also compared the IMAT plan with an IMRT plan for the RPC phantom. RESULTS: All plans that incorporated RIF and LPO had lower objective function values than those that incorporated LPO only. The objective function value was reduced by about 15% after the generation of RIF arcs and 52% after generation of RIF arcs and leaf position optimization. The IMAT plan for the RPC phantom had similar dose coverage for PTV1 and PTV2 (the same dose volume histogram curves), however, slightly lower dose to the normal tissues compared to a six-field IMRT plan. CONCLUSION: The use of a ray importance factor can generate initial IMAT arcs efficiently for further MLC leaf position optimization to obtain more favorable IMAT plan.

Commissioning a fast Monte Carlo dose calculation algorithm for lung cancer treatment planning.

A commercial Monte Carlo simulation package, NXEGS 1.12 (NumeriX LLC, New York, NY), was commissioned for photon-beam dose calculations. The same sets of measured data from 6-MV and 18-MV beams were used to commission NXEGS and Pinnacle 6.2b (Philips Medical Systems, Andover, MA). Accuracy and efficiency were compared against the collapsed cone convolution algorithm implemented in Pinnacle 6.2b, together with BEAM simulation (BEAMnrc 2001: National Research Council of Canada, Ottawa, ON). We investigated a number of options in NXEGS: the accuracy of fast Monte Carlo, the re-implementation of EGS4, post-processing technique (dose de-noising algorithm), and dose calculation time. Dose distributions were calculated with NXEGS, Pinnacle, and BEAM in water, lung-slab, and air-cylinder phantoms and in a lung patient plan. We compared the dose distributions calculated by NXEGS, Pinnacle, and BEAM. In a selected region of interest (7725 voxels) in the lung phantom, all but 1 voxel had a gamma (3% and 3 mm thresholds) of 1 or less for the dose difference between the NXEGS re-implementation of EGS4 and BEAM, and 99% of the voxels had a gamma of 1 or less for the dose difference between NXEGS fast Monte Carlo and BEAM. Fast Monte Carlo with post-processing was up to 100 times faster than the NXEGS re-implementation of EGS4, while maintaining +/- 2% statistical uncertainty. With air inhomogeneities larger than 1 cm, post-processing preserves the dose perturbations from the air cylinder. When 3 or more beams were used, fast Monte Carlo with post-processing was comparable to or faster than Pinnacle 6.2b collapsed cone convolution.

Monte Carlo dose calculation of segmental IMRT delivery to a moving phantom using dynamic MLC and gating log files.

Respiratory gating is emerging as a tool to limit the effect of motion for liver and lung tumors. In order to study the impact of target motion and gated intensity modulated radiation therapy (IMRT) delivery, a computer program was developed to simulate segmental IMRT delivery to a moving phantom. Two distinct plans were delivered to a rigid-motion phantom with a film insert in place under four conditions: static, sinusoidal motion, gated sinusoidal motion with a duty cycle of 25% and gated sinusoidal motion with duty cycle of 50% under motion conditions of a typical patient (A = 1 cm, T = 4 s). The MLC controller log files and gating log files were retained to perform a retrospective Monte Carlo dose calculation of the plans. Comparison of the 2D planar dose distributions between simulation and measurement demonstrated that our technique had at least 94% of the points passing gamma criteria of 3% for dose difference and 3 mm as the distance to agreement. This note demonstrates that the use of dynamic multi-leaf collimator and respiratory monitoring system log files together with a fast Monte Carlo dose calculation algorithm is an accurate and efficient way to study the dosimetric effect of motion for gated or non-gated IMRT delivery on a rigidly-moving body.

Segmentation and leaf sequencing for intensity modulated arc therapy.

A common method in generating intensity modulated radiation therapy (IMRT) plans consists of a three step process: an optimized fluence intensity map (IM) for each beam is generated via inverse planning, this IM is then segmented into discrete levels, and finally, the segmented map is translated into a set of MLC apertures via a leaf sequencing algorithm. To date, limited work has been done on this approach as it pertains to intensity modulated arc therapy (IMAT), specifically in regards to the latter two steps. There are two determining factors that separate IMAT segmentation and leaf sequencing from their IMRT equivalents: (1) the intrinsic 3D nature of the intensity maps (standard 2D maps plus the angular component), and (2) that the dynamic multileaf collimator (MLC) constraints be met using a minimum number of arcs. In this work, we illustrate a technique to create an IMAT plan that replicates Tomotherapy deliveries by applying IMAT specific segmentation and leaf-sequencing algorithms to Tomotherapy output sinograms. We propose and compare two alternative segmentation techniques, a clustering method, and a bottom-up segmentation method (BUS). We also introduce a novel IMAT leaf-sequencing algorithm that explicitly takes leaf movement constraints into consideration. These algorithms were tested with 51 angular projections of the output leaf-open sinograms generated on the Hi-ART II treatment planning system (Tomotherapy Inc.). We present two geometric phantoms and 2 clinical scenarios as sample test cases. In each case 12 IMAT plans were created, ranging from 2 to 7 intensity levels. Half were generated using the BUS segmentation and half with the clustering method. We report on the number of arcs produced as well as differences between Tomotherapy output sinograms and segmented IMAT intensity maps. For each case one plan for each segmentation method is chosen for full Monte Carlo dose calculation (NumeriX LLC) and dose volume histograms (DVH) are calculated. In all cases, the BUS method outperformed the clustering, method. We recommend using the BUS algorithm and discuss potential improvements to the clustering algorithms.

A treatment planning study comparing whole breast radiation therapy against conformal, IMRT and tomotherapy for accelerated partial breast irradiation.

PURPOSE AND BACKGROUND: Conventional early breast cancer treatment consists of a lumpectomy followed by whole breast radiation therapy. Accelerated partial breast irradiation (APBI) is an investigational approach to post-lumpectomy radiation for early breast cancer. The purpose of this study is to compare four external beam APBI techniques, including tomotherapy, with conventional whole breast irradiation for their radiation conformity index, dose homogeneity index, and dose to organs at risk. METHODS AND MATERIALS: Small-field tangents, three-dimensional conformal radiation therapy, intensity-modulated radiation therapy and helical tomotherapy were compared for each of 15 patients (7 right, 8 left). One radiation conformity and two dose homogeneity indices were used to evaluate the dose to the target. The mean dose to organs at risk was also evaluated. RESULTS: All proposed APBI techniques improved the conformity index significantly over whole breast tangents while maintaining dose homogeneity and without a significant increase in dose to organs at risk. CONCLUSION: The four-field IMRT plan produced the best dosimetric results; however this technique would require appropriate respiratory motion management. An alternative would be to use a four-field conformal technique that is less sensitive to the effects of respiratory motion.

AFM study of forces between silica, silicon nitride and polyurethane pads.

Interaction of silica and silicon nitride with polyurethane surfaces is rather poorly studied despite being of great interest for modern semiconductor industry, e.g., for chemical-mechanical planarization (CMP) processes. Here we show the results from the application of the atomic force microscopy (AFM) technique to study the forces between silica or silicon nitride (AFM tips) and polyurethane surfaces in aqueous solutions of different acidity. The polyurethane surface potentials are derived from the measured AFM data. The obtained potentials are in rather good agreement with measurements of zeta-potentials using the streaming-potentials method. Another important parameter, adhesion, is also measured. While the surface potentials of silica are well known, there are ambiguous results on the potentials of silicon nitride that is naturally oxidized. Deriving the surface potential of the naturally oxidized silicon nitride from our measurements, we show that it is not oxidized to silica despite some earlier published expectations.

Preparation of planar retinal specimens: verification by histology, mRNA profiling, and proteome analysis.

PURPOSE: Elucidation of the transcriptome and proteome of the normal retina will be difficult since it is comprised of at least 55 different cell types. However the characteristic layered cellular anatomy of the retina makes it amenable to planar sectioning, enabling the generation of enriched retinal cell populations. The aim of this study was to validate a reproducible method for preparing enriched retinal layers from porcine retina. METHODS: The thicknesses of the retinal photoreceptor, inner nuclear and ganglion cell, and fiber layers were determined by routine histology of cross sections of fresh whole retina mounted on polyvinylidene difluoride (PVDF) membrane. Dissected retina (5 mm2) was placed on PVDF membrane and a series of planar cryosections corresponding to the photoreceptor and inner nuclear layer were removed leaving the ganglion cell and fiber layer which was subsequently detached from the membrane. The retinal specimens were stored at -80 degrees C. Representative planar tissue sections were sonicated in ice-chilled 40 mM ammonium bicarbonate pH 7.9 and aliquots removed for RNA extraction. Quantitative RT-PCR was used to analyze the mRNA expression of genes indicative of specific retinal layers. Ammonium bicarbonate protein extracts were centrifuged, lyophilized and prepared for direct liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using a Waters Q-Tof Ultima. RESULTS: Histological analysis established the parameters for planar cryosectioning: photoreceptor layer (69+/-1.8 microm), outer plexiform (11+/-0.6 microm), inner nuclear layer (28+/-0.5 microm), inner plexiform, ganglion cell and fiber layer (100+/-5.3 microm). Gene expression profiling provided an independent method for validating the respective retinal preparations. For example, glial fibrillary acidic protein (GFAP) was expressed up to 21 fold higher in the inner retinal "ganglion cell enriched" fraction than in the outer retinal "photoreceptor enriched" fraction. The pattern was reversed for blue cone opsin, which was expressed up to 24 fold higher in the "photoreceptor enriched" fraction. Endogenous protein fragments indicative of each layer were identified by mass spectrometry and de novo sequence data obtained. CONCLUSIONS: Combined histological and mRNA expression profiling has confirmed the development of a reproducible method for generating validated porcine retinal layers enriched for specific cell types. Direct proteome analysis detected endogenous peptide fragments of characteristic retinal proteins. Further analysis of these enriched retinal cell preparations will facilitate a more selective investigation of the retinal transcriptome and proteome than studies of the intact retina.