Inter-operator Precision Research Articles

B-361 Novel Blood-based Assay for Early Cancer Detection Demonstrates High Precision and Reproducibility on Low Tumor Content

Abstract Background Early detection of cancer has significant potential to impact human health and society by decreasing cancer-related morbidity and mortality. While previous approaches to identify cancer-informative biomarkers are predominantly statistical, Harbinger Health has utilized foundational discoveries from developmental biology to design a targeted methylation assay for early cancer detection from cell-free DNA (cfDNA) extracted from plasma. Utilizing this biologically informed approach, we developed a fixed multi-layered logistic regression-based machine learning algorithm, trained with an in-house generated dataset of 1046 samples (621 cancer, 425 non-cancer) that predicts a binary classification (yes/no) for cfDNA samples processed through our assay. We have previously reported high sensitivity for multi-cancer detection, including for early-stage disease. Methods Here, we perform a comprehensive independent analytical validation of our assay and algorithm, encompassing 69 subjects: 19 with newly diagnosed treatment-naïve cancer (8 different cancer types) and 50 individuals with no history, diagnosis, or cancer symptoms. In total, we utilized 122 replicate samples to assess reproducibility and precision, 8 non-template controls (water) to determine limit of blank (LOB), and cohorts of matched biopsy and cfDNA to determine tumor content limit of detection (LOD). Results Precision was assessed within five different sub-studies, by comparing concordance of predicted binary cancer classification between replicate samples, giving results of 0.90 (0.95 CI: 0.764–0.959) for inter-run precision, 1.00 (0.95 CI: 0.796–1.000) for intra-run precision, 1.00 (0.95 CI: 0.871–1.000) for inter-operator precision, 0.96 (0.95 CI: 0.930–0.998) for inter-instrument precision and 0.83 (0.95 CI: 0.641–0.933) for inter-day precision. To determine LOB, we carried 8 non-template controls (water) through the entire assay and detected on average ∼0.02% unique aligned reads of a true sample on the same sequencing run. Finally, to assess tumor content LOD, we developed methodology that uses methylation signal to estimate the amount of tumor-derived DNA in each cfDNA sample and validated our estimates using whole exome sequencing, an orthogonal gold-standard approach. We then assessed the relationship between tumor content and classifier sensitivity using our training data of 625 cancer cfDNA samples and determined that our tumor content LOD whereby 95% true cancer samples were correctly predicted to be 0.037%. Conclusion Our assay shows high performance and high technical reproducibility. Our previously reported high sensitivity for stage 1 and stage 2 cancers, as well as extremely low tumor content LOD reported here supports our ability to perform early-stage multi-cancer detection, where the levels of circulating tumor DNA are low.

Evaluation of two methods of bone age assessment in peripubertal children in Zimbabwe.

Bone age (BA) measurement in children is used to evaluate skeletal maturity and helps in the diagnosis of growth disorders in children. The two most used methods are Greulich and Pyle (GP), and Tanner and Whitehouse 3 (TW3), both based upon assessment of a hand-wrist radiograph. To our knowledge no study has compared and validated the two methods in sub-Saharan Africa (SSA), and only a few have determined BA despite it being a region where skeletal maturity is often impaired for example by HIV and malnutrition. This study aimed to compare BA as measured by two methods (GP and TW3) against chronological age (CA) and determine which method is most applicable in peripubertal children in Zimbabwe. We conducted a cross-sectional study of boys and girls who tested negative for HIV. Children and adolescents were recruited by stratified random sampling from six schools in Harare, Zimbabwe. Non-dominant hand-wrist radiographs were taken, and BA assessed manually using both GP and TW3. Paired sample Student t-tests were used to calculate the mean differences between BA and chronological age (CA) in boys and girls. Bland-Altman plots compared CA to BA as determined by both methods, and agreement between GP and TW3 BA. All radiographs were graded by a second radiographer and 20% of participants of each sex were randomly selected and re-graded by the first observer. Intraclass correlation coefficient assessed intra- and inter-rater reliability and coefficient of variation assessed precision. We recruited 252 children (111 [44%] girls) aged 8.0-16.5years. The boys and girls were of similar mean±SD CA (12.2±2.4 and 11.7±1.9years) and BA whether assessed by GP (11.5±2.8 and 11.5±2.1years) or TW3 (11.8±2.5 and 11.8±2.1years). In boys BA was lower than CA by 0.76years (95% CI: -0.95, -0.57) when using GP, and by 0.43years (95% CI: -0.61, -0.24) when using TW3. Among the girls there was no difference between BA and CA by either GP [-0.19years (95% CI: -0.40, 0.03)] or TW3 [0.07years (95% CI: -0.16, 0.29)]. In both boys and girls, there were no systematic differences between CA and TW3 BA across age groups whereas agreement improved between CA and GP BA as children got older. Inter-operator precision was 1.5% for TW3 and 3.7% for GP (n=252) and intra-operator precision was 1.5% for TW3 and 2.4% for GP (n=52). The TW3 BA method had better precision than GP and did not systematically differ from CA, meaning that TW3 is the preferred method of assessment of skeletal maturity in Zimbabwean children and adolescents. TW3 and GP methods do not agree for estimates of BA and therefore cannot be used interchangeably. The systematic differences in GP BA assessments over age means it is not appropriate for use in all age groups or stages of maturity in this population.

Optimisation and Validation of a conventional ELISA and cut-offs for detecting and quantifying anti-SARS-CoV-2 Spike, RBD, and Nucleoprotein IgG, IgM, and IgA antibodies in Uganda.

There is an urgent need for better immunoassays to measure antibody responses as part of immune-surveillance activities and to profile immunological responses to emerging SARS-CoV-2 variants. We optimised and validated an in-house conventional ELISA to identify and quantify SARS-CoV-2 spike- (S-), receptor binding domain- (RBD-), and nucleoprotein- (N-) directed IgG, IgM, and IgA binding antibodies in the Ugandan population and similar settings. Pre- and post-pandemic specimens were used to compare the utility of mean ± 2SD, mean ± 3SD, 4-fold above blanks, bootstrapping, and receiver operating characteristic (ROC) analyses in determining optimal cut-off optical densities at 450 nm (OD) for discriminating between antibody positives and negatives. "Limits of detection" (LOD) and "limits of quantitation" (LOQ) were validated alongside the assay's uniformity, accuracy, inter-assay and inter-operator precision, and parallelism. With spike-directed sensitivity and specificity of 95.33 and 94.15%, respectively, and nucleoprotein sensitivity and specificity of 82.69 and 79.71%, ROC was chosen as the best method for determining cutoffs. Accuracy measurements were within the expected CV range of 25%. Serum and plasma OD values were highly correlated (r = 0.93, p=0.0001). ROC-derived cut-offs for S-, RBD-, and N-directed IgG, IgM, and IgA were 0.432, 0.356, 0.201 (S), 0.214, 0.350, 0.303 (RBD), and 0.395, 0.229, 0.188 (N). The sensitivity and specificity of the S-IgG cut-off were equivalent to the WHO 20/B770-02 S-IgG reference standard at 100% level. Spike negative IgG, IgM, and IgA ODs corresponded to median antibody concentrations of 1.49, 3.16, and 0 BAU/mL, respectively, consistent with WHO low titre estimates. Anti-spike IgG, IgM, and IgA cut-offs were equivalent to 18.94, 20.06, and 55.08 BAU/mL. For the first time, we provide validated parameters and cut-off criteria for the in-house detection of subclinical SARS-CoV-2 infection and vaccine-elicited binding antibodies in the context of Sub-Saharan Africa and populations with comparable risk factors.

“TEG” talks: technology worth spreading?

“TEG” talks: technology worth spreading?

Analytical validity of DecisionDx-SCC, a gene expression profile test to identify risk of metastasis in cutaneous squamous cell carcinoma (SCC) patients

BackgroundTo improve identification of patients with cutaneous squamous cell carcinoma (SCC) at high risk for metastatic disease, the DecisionDx-SCC assay, a prognostic 40-gene expression profile (40-GEP) test, was developed and validated. The 40-GEP assay utilizes RT-PCR gene expression analysis on primary tumor biopsy tissue to evaluate the expression of 34 signature gene targets and 6 normalization genes. The test provides classifications of low risk (Class 1), moderate risk (Class 2A), and high risk (Class 2B) of metastasis within 3 years of diagnosis. The primary objective of this study was to validate the analytical performance of the 40-gene expression signature.MethodsThe repeatability and reproducibility of the 40-GEP test was evaluated by performance of inter-assay, intra-assay, and inter-operator precision experiments along with monitoring the reliability of sample and reagent stability for class call concordance. The technical performance of clinical orders from September 2020 through July 2021 for the 40-GEP test was assessed.ResultsPatient hematoxylin and eosin (H&E) stained slides were reviewed by a board-certified pathologist to assess minimum acceptable tumor content. Class specific controls (Class 1 and Class 2B) were evaluated with Levey-Jennings analysis and demonstrated consistent and reproducible results. Inter-assay, inter-operator and intra-assay concordance were all ≥90%, with short-term and long-term RNA stability also meeting minimum concordance requirements. Of the 2586 orders received, 93.5% remained eligible for testing, with 97.1% of all tested samples demonstrating actionable class call results.ConclusionDecisionDx-SCC demonstrates a high degree of analytical precision, yielding high concordance rates across multiple performance experiments, along with exhibiting robust technical reliability on clinical samples.

Abstract 1308: Development and validation of a flow cytometric assay for measuring glycoprotein A repetitions predominant protein GARP target engagement by ABBV-151

Abstract Introduction: GARP is a cell surface protein expressed post activation on regulatory T-cells (Treg), monocytes, B cells, and platelets. GARP binds to latent transforming growth factor beta (TGF-β1) and regulates the release of active TGF-β1. TGF-β1 modulates Treg-mediated immunosuppression and represses effector T cell responses. ABBV-151 is a first-in-class monoclonal antibody that binds to the GARP-TGF-β1 complex and blocks active TGF-β1 release. Here we describe the validation of a TE assay for ABBV-151 using human platelets to measure receptor occupancy (RO) and binding saturation, that will inform dosing decisions during a first-in-human Phase I dose escalation study. Procedure: A competing (LHG10.6 that recognizes the GARP-TGF-β1 complex) and non-competing (1E7 that recognizes total GARP) anti-GARP antibodies were used to measure TE on platelets. Human whole blood was incubated with increasing concentrations of ABBV-151, followed by platelet-rich plasma (PRP) preparation and storage at -80°C. Platelets were thawed, washed, and stained with a fluorescently labeled antibody cocktail (LHGH10.6, 1E7 and CD61) and analyzed on a flow cytometer. The analytical measurement range, inter-operator and inter-assay precision and sample stability at -80°C (up to 12 months) for this TE assay was determined. Results: ABBV-151 TE assay demonstrated a dose response relationship over a range of ABBV-151 concentrations (0.08 to 400 ng/mL) that corresponded to 0-80% occupancy, with an EC50 of 6.2 ± 2.1 ng/mL, and 0-100% saturation with an EC50 of 6.7 ± 1.9 ng/mL. Inter-assay precision demonstrated ≤3% CV at low ABBV-151 doses, and ≤14% in samples at high doses. Inter-operator precision for untreated and treated samples was between 3 to 9% CV, respectively. The stability data indicated frozen platelets could be stored up to 12 months with highly reproducible assay performance. Conclusions: This TE assay for ABBV-151 on platelets demonstrates desirable analytical performance characteristics. The percent saturation (PS) showed higher precision than the RO measurements for this assay and PS was determined to be a reliable metric for measuring TE. Frozen platelets samples were suitable for the TE assay for up to a year allowing for batch processing of samples at a central lab and for minimizing the variability in test results. This novel TE assay overcomes key limitations of implementing TE assays in clinical trials, and quantification of PS using this TE assay in clinical trials is currently ongoing to assess the pharmacodynamic/pharmacokinetic relationship of ABBV-151 therapy. Citation Format: Juan M. Cardenas, Jayaprakash Kotha, Sridevi Kotha, Lisa K. Jennings, Michelle Graham, Stacie Lambert, Kinjal Hew. Development and validation of a flow cytometric assay for measuring glycoprotein A repetitions predominant protein GARP target engagement by ABBV-151 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1308.

An Assay to Monitor the Engagement and Modulation of CD6 on T cells as a Clinical Biomarker of Treatment with Itolizumab

Abstract Itolizumab is a novel first-in-class monoclonal antibody that selectively targets the co-stimulatory molecule CD6, a receptor that is highly expressed on CD4 and CD8 T cells and plays an important role in activation and migration. Monitoring target engagement and changes in receptor levels is critically important to interpreting clinical data. To evaluate the pharmacodynamic properties of itolizumab treatment on T cells in patients including those with graft versus host disease (GvHD), Precision for Medicine has developed and validated a 10-color flow cytometry assay to assess the engagement and modulation of cell-surface CD6. The assay was validated using whole blood from healthy donors. Blood was spiked with five concentrations of drug selected based on expected PK in the clinical study. The assay conditions were optimized for sensitivity, signal: noise ratio, detection of free receptor, receptor-bound itolizumab and total surface CD6. Itolizumab was detected using a fluorochrome conjugated α-human IgG1 antibody while total surface CD6 expression was assessed using an antibody to a non-competing site on CD6. For validation, pre-set criteria were used to assess inter-assay, intra-assay, inter-operator precision and post-staining stability. Technical validation was successfully met; and the assay performs within acceptable precision parameters. Measuring cell-based receptor engagement and fate in patients on immuno-modulatory therapies is very challenging. This assay was designed and validated to be both sensitive and selective in the quantification of CD6 receptor occupancy and modulation to facilitate the determination of an optimal therapeutic dose in autoimmune and inflammatory diseases.

Analyzing Femorotibial Cartilage Thickness Using Anatomically Standardized Maps: Reproducibility and Reference Data.

Alterations in cartilage thickness (CTh) are a hallmark of knee osteoarthritis, which remain difficult to characterize at high resolution, even with modern magnetic resonance imaging (MRI), due to a paucity of standardization tools. This study aimed to assess a computational anatomy method producing standardized two-dimensional femorotibial CTh maps. The method was assessed with twenty knees, processed following three common experimental scenarios. Cartilage thickness maps were obtained for the femorotibial cartilages by reconstructing bone and cartilage mesh models in tree-dimension, calculating three-dimensional CTh maps, and anatomically standardizing the maps. The intra-operator accuracy (median (interquartile range, IQR) of −0.006 (0.045) mm), precision (0.152 (0.070) mm), entropy (7.02 (0.71) and agreement (0.975 (0.020))) results suggested that the method is adequate to capture the spatial variations in CTh and compare knees at varying osteoarthritis stages. The lower inter-operator precision (0.496 (0.132) mm) and agreement (0.808 (0.108)) indicate a possible loss of sensitivity to detect differences in a setting with multiple operators. The results confirmed the promising potential of anatomically standardized maps, with the lower inter-operator reproducibility stressing the need to coordinate operators. This study also provided essential reference data and indications for future research using CTh maps.

A new method for quantitative assessment of hand muscle volume and fat in magnetic resonance images

BackgroundRheumatoid arthritis (RA) is characterized by systemic inflammation and bone and muscle loss. Recent research showed that obesity facilitates inflammation, but it is unknown if obesity also increases the risk or severity of RA. Further research requires an accurate quantification of muscle volume and fat content.MethodsThe aim was to develop a reproducible (semi) automated method for hand muscle segmentation and quantification of hand muscle fat content and to reduce the time consuming efforts of manual segmentation. T1 weighted scans were used for muscle segmentation based on a random forest classifier. Optimal segmentation parameters were determined by cross validation with 30 manually segmented hand datasets (gold standard). An operator reviewed the automatically created segmentation and applied corrections if necessary. For fat quantification, the segmentation masks were automatically transferred to MRI Dixon sequences by rigid registration. In total 76 datasets from RA patients were analyzed. Accuracy was validated against the manual gold standard segmentations.ResultsAverage analysis time per dataset was 10 min, more than 10 times faster compared to manual outlining. All 76 datasets could be analyzed and were accurate as judged by a clinical expert. 69 datasets needed minor manual segmentation corrections. Segmentation accuracy compared to the gold standard (Dice ratio 0.98 ± 0.04, average surface distance 0.04 ± 0.10 mm) and reanalysis precision were excellent. Intra- and inter-operator precision errors were below 0.3% (muscle) and 0.7% (fat). Average Hausdorff distances were higher (1.09 mm), but high values originated from a shift of the analysis VOI by one voxel in scan direction.ConclusionsWe presented a novel semi-automated method for quantitative assessment of hand muscles with excellent accuracy and operator precision, which highly reduced a traditional manual segmentation effort. This method may greatly facilitate further MRI image based muscle research of the hands.

Optimizing HR-pQCT workflow: a comparison of bias and precision error for quantitative bone analysis.

High-resolution peripheral quantitative computed tomography workflow includes manually correcting contours generated by the manufacturer's automated protocol. There is interest in minimizing corrections to save time and reduce precision error; however, bias related to the automated protocol is unknown. This study quantifies error bias when contours are uncorrected and identifies the impact of operator training on bias and precision error. Forty-five radii and tibiae scans across a representative range of bone density were analyzed using the automated and manually corrected contours of three operators, with training ranging from beginner to expert, and compared with a "ground truth" to estimate bias. Inter-operator precision was measured across operators. The tibia had greater error bias than the radius when contours were uncorrected, with compartmental bone mineral densities and cortical microarchitecture having greatest biases, which could have significant implications for interpretation of studies using this skeletal site. Bias and precision error were greatest when contours were corrected by the beginner operator; however, when this operator was removed, bias was no longer present and inter-operator precision was between 0.01 and 3.74% for all parameters except cortical porosity. These findings establish the need for manual correction and provide guidance on operator training needed to maximize workflow efficiency.

Abstract 2299: Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients

Abstract Introduction The presence of MRD in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 (FLT3) internal tandem duplications (ITD) confer a negative prognostic impact by increasing risk of relapse. The ability to detect FLT3-ITD mutations in remission bone marrow specimens is hampered by the limited sensitivity at 1% of PCR-based assays. To address such limitations, we developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations. Method Genomic DNA was isolated from bone marrow (BM) aspirates or peripheral blood (PB) samples. PCR was performed to amplify exons 13 to 15 of the FLT3 gene. Highly diverse NGS libraries were then generated and sequenced using Illumina’s sequencer. Using a custom bioinformatics approach, unique FLT3-ITD mutations of varying lengths were identified and mutant allelic frequency calculated. The assay was validated using clinical samples and contrived samples. For accuracy assessment, 30 samples (including PB or BM remnant patient DNA and cell line DNA, above DNA diluted in normal DNA) were included. Data were compared with a Fragment Size Analysis by Capillary Electrophoresis assay. To assess precision, the assay was validated at multiple levels evaluating intra-assay, inter-assay, inter-operator, inter-instrument and inter-reagent lot precision. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. Summary The ideal DNA input range was established as 300 ng to 500 ng. In all FLT3-ITD-positive cell line samples covering diverse FLT3-ITD lengths (6 bp to 156 bp), the FLT3-ITD MRD NGS assay showed 100% concordance with the reference assay. The assay is also capable of detecting ITDs as expected after the samples were diluted in normal DNA to mimic samples from patients in remission. All acceptance criteria for the different precision parameters were met. The lower limit of detection of 0.013% regardless of ITD length was established but the assay was capable of detecting FLT3-ITD mutations at a level as low as 0.003% without false-positive results. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% or the lower limit of quantitation. Conclusion Analytical validation results established the role of this NGS-based MRD assay for the clinical management of FLT3-ITD AML. The FLT3-ITD MRD NGS assay demonstrated high sensitivity and high specificity by detecting the unique length of each patient’s mutation with no false-positive results in expected negative samples. This assay might be helpful in defining the depth of remission, identifying persistent disease, and helping to guide decision making in the use of FLT3 inhibitors as continuation therapy. Citation Format: Wenge Shi, Christian Laing, Wei Ding, Marc Mycoco, Jelveh Lameh, Reinhold Pollner. Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2299.

Phantomless calibration of CT scans for measurement of BMD and bone strength—Inter-operator reanalysis precision

Patient-specific phantomless calibration of computed tomography (CT) scans has the potential to simplify and expand the use of pre-existing clinical CT for quantitative bone densitometry and bone strength analysis for diagnostic and monitoring purposes. In this study, we quantified the inter-operator reanalysis precision errors for a novel implementation of patient-specific phantomless calibration, using air and either aortic blood or hip adipose tissue as internal calibrating reference materials, and sought to confirm the equivalence between phantomless and (traditional) phantom-based measurements. CT scans of the spine and hip for 25 women and 15 men (mean±SD age of 67±9years, range 41–86years), one scan per anatomic site per patient, were analyzed independently by two analysts using the VirtuOst software (O.N. Diagnostics, Berkeley, CA). The scans were acquired at 120kVp, with a slice thickness/increment of 3mm or less, on nine different CT scanner models across 24 different scanners. The main parameters assessed were areal bone mineral density (BMD) at the hip (total hip and femoral neck), trabecular volumetric BMD at the spine, and vertebral and femoral strength by finite element analysis; other volumetric BMD measures were also assessed. We found that the reanalysis precision errors for all phantomless measurements were ≤0.5%, which was as good as for phantom calibration. Regression analysis indicated equivalence of the phantom- versus phantomless-calibrated measurements (slope not different than unity, R2≥0.98). Of the main parameters assessed, non-significant paired mean differences (n=40) between the two measurements ranged from 0.6% for hip areal BMD to 1.1% for mid-vertebral trabecular BMD. These results indicate that phantom-equivalent measurements of both BMD and finite element-derived bone strength can be reliably obtained from CT scans using patient-specific phantomless calibration.

A reproducible semi-automatic method to quantify the muscle-lipid distribution in clinical 3D CT images of the thigh.

Many studies use threshold-based techniques to assess in vivo the muscle, bone and adipose tissue distribution of the legs using computed tomography (CT) imaging. More advanced techniques divide the legs into subcutaneous adipose tissue (SAT), anatomical muscle (muscle tissue and adipocytes within the muscle border) and intra- and perimuscular adipose tissue. In addition, a so-called muscle density directly derived from the CT-values is often measured. We introduce a new integrated approach to quantify the muscle-lipid system (MLS) using quantitative CT in patients with sarcopenia or osteoporosis. The analysis targets the thigh as many CT studies of the hip do not include entire legs The framework consists of an anatomic coordinate system, allowing delineation of reproducible volumes of interest, a robust semi-automatic 3D segmentation of the fascia and a comprehensive method to quantify of the muscle and lipid distribution within the fascia. CT density-dependent features are calibrated using subject-specific internal CT values of the SAT and external CT values of an in scan calibration phantom. Robustness of the framework with respect to operator interaction, image noise and calibration was evaluated. Specifically, the impact of inter- and intra-operator reanalysis precision and addition of Gaussian noise to simulate lower radiation exposure on muscle and AT volumes, muscle density and 3D texture features quantifying MLS within the fascia, were analyzed. Existing data of 25 subjects (age: 75.6 ± 8.7) with porous and low-contrast muscle structures were included in the analysis. Intra- and inter-operator reanalysis precision errors were below 1% and mostly comparable to 1% of cohort variation of the corresponding features. Doubling the noise changed most 3D texture features by up to 15% of the cohort variation but did not affect density and volume measurements. The application of the novel technique is easy with acceptable processing time. It can thus be employed for a comprehensive quantification of the muscle-lipid system enabling radiomics approaches to musculoskeletal disorders.

Precision and sources of variability in the assessment of rheumatoid arthritis erosions by HRpQCT

ObjectivesTo assess the precision and the sources of variation due to repositioning of the manual measurement of erosions located on the metacarpophalangeal joints (MCP) with High-Resolution peripheral Quantitative Computed Tomography (HRpQCT) in rheumatoid arthritis (RA). MethodsRA patients with at least one erosion on the 2nd, 3rd or 4th MCP on conventional radiographs were included. Two scans were performed the same day with repositioning. The main outcome was to calculate the short-term precision of the width, depth, and volume of erosions. Secondary outcomes were intra-operator and inter-operator precision, the least significant change, and the sources of variability of the measurement. ResultsTwenty-nine patients were included, allowing analysis of 406 erosions from 0.9 to 3mm of diameter. Intraclass correlation coefficients (ICC) for the precision of the measurement of the axial width, axial depth, and volume after repositioning were 0.80, 0.96, and 0.99, respectively. RMS CV and RMS SD were 16%, 0.26mm; 17.5%, 0.32mm; and 19.7%, 0.93mm3, respectively. For intra-operator precision, ICCs were 0.92, 0.97, and 0.99 with RMS CV of 16%, 16.4%, and 18.7%, respectively. Inter-operator precision of the volume was 0.99 with RMS CV of 14%. Least significant change of width, depth, and volume were 0.3mm, 0.2mm, and 0.3mm3. There was no significant correlation with bone microarchitecture parameters. ConclusionHRpQCT analysis is a reproducible method to characterize and measure erosions, without effect of the repositioning. However, we showed weak precision in manual measurement due to intra-operator variability.

Operator variability in scan positioning is a major component of HR-pQCT precision error and is reduced by standardized training.

HR-pQCT is increasingly used to assess bone quality, fracture risk, and anti-fracture interventions. The contribution of the operator has not been adequately accounted in measurement precision. Operators acquire a 2D projection ("scout view image") and define the region to be scanned by positioning a "reference line" on a standard anatomical landmark. In this study, we (i) evaluated the contribution of positioning variability to in vivo measurement precision, (ii) measured intra- and inter-operator positioning variability, and (iii) tested if custom training software led to superior reproducibility in new operators compared to experienced operators. To evaluate the operator in vivo measurement precision, we compared precision errors calculated in 64 co-registered and non-co-registered scan-rescan images. To quantify operator variability, we developed software that simulates the positioning process of the scanner's software. Eight experienced operators positioned reference lines on scout view images designed to test intra- and inter-operator reproducibility. Finally, we developed modules for training and evaluation of reference line positioning. We enrolled six new operators to participate in a common training, followed by the same reproducibility experiments performed by the experienced group. In vivo precision errors were up to threefold greater (Tt.BMD and Ct.Th) when variability in scan positioning was included. The inter-operator precision errors were significantly greater than the short-term intra-operator precision (p < 0.001). New trained operators achieved comparable intra-operator reproducibility to experienced operators and lower inter-operator reproducibility (p < 0.001). Precision errors were significantly greater for the radius than for the tibia. Operator reference line positioning contributes significantly to in vivo measurement precision and is significantly greater for multi-operator datasets. Inter-operator variability can be significantly reduced using a systematic training platform, now available online ( http://webapps.radiology.ucsf.edu/refline/ ).

Guided mass spectrum labelling in atom probe tomography

Atom probe tomography (APT) is a valuable near-atomic scale imaging technique, which yields mass spectrographic data. Experimental correctness can often pivot on the identification of peaks within a dataset, this is a manual process where subjectivity and errors can arise. The limitations of manual procedures complicate APT experiments for the operator and furthermore are a barrier to technique standardisation. In this work we explore the capabilities of computer-guided ranging to aid identification and analysis of mass spectra.We propose a fully robust algorithm for enumeration of the possible identities of detected peak positions, which assists labelling. Furthermore, a simple ranking scheme is developed to allow for evaluation of the likelihood of each possible identity being the likely assignment from the enumerated set. We demonstrate a simple, yet complete work-chain that allows for the conversion of mass-spectra to fully identified APT spectra, with the goal of minimising identification errors, and the inter-operator variance within APT experiments.This work chain is compared to current procedures via experimental trials with different APT operators, to determine the relative effectiveness and precision of the two approaches. It is found that there is little loss of precision (and occasionally gain) when participants are given computer assistance. We find that in either case, inter-operator precision for ranging varies between 0 and 2 “significant figures” (2σ confidence in the first n digits of the reported value) when reporting compositions. Intra-operator precision is weakly tested and found to vary between 1 and 3 significant figures, depending upon species composition levels. Finally it is suggested that inconsistencies in inter-operator peak labelling may be the largest source of scatter when reporting composition data in APT.

Segmentation and quantification of bone erosions in high-resolution peripheral quantitative computed tomography datasets of the metacarpophalangeal joints of patients with rheumatoid arthritis

To develop a precise three-dimensional (3D) segmentation technique for bone erosions in high-resolution peripheral quantitative CT (HR-pQCT) datasets to measure their volume, surface area and shape parameters. Assessment of bone erosions in patients with RA is important for diagnosis and evaluation of treatment efficacy. HR-pQCT allows quantifying periarticular bone loss in arthritis. HR-pQCT scans with a spatial resolution of about 120 µm of the second to fourth metacarpophalangeal joints were acquired in patients with RA. Erosions were identified by placing a seed point in each of them. After applying 3D segmentation, the volume, surface area and sphericity of erosions were calculated. Results were compared with an approximation method using manual measurements. Intra- and interoperator precision analysis was performed for both the 3D segmentation and the manual technique. Forty-three erosions were assessed in 18 datasets. Intra- and interoperator precisions (RMSCV/RMSSD) for erosion volume were 5.66%/0.49 mm(3) and 7.76%/0.76 mm(3), respectively. The correlation between manual measurements and their simulation using segmentation volumes was r = 0.87. Precision errors for the manual method were 15.39% and 0.36 mm(3), respectively. We developed a new precise 3D segmentation technique for quantification of bone erosions in HR-pQCT datasets that correlates to the volume, shape and surface area of the erosion. The technique allows fast and effective measurement of the erosion size and could therefore be helpful for rapid and quantitative assessment of erosion size.

Application of sub-regional analysis to bone mineral density of the lower limb from whole body DXA scans

Bone mineral density at spine and hip is widely used to diagnose osteoporosis. Certain conditions cause changes in bone density at other sites, particularly in the lower limb, with fractures occurring in non-classical locations. Bone density changes at these sites would be of interest for diagnosis and treatment. We describe an application, based on an existing software option for Hologic scanners, which allows reproducible measurement of bone density at six lower limb sites (upper femur, mid-femur, lower femur; upper leg, mid-leg, lower leg). In 30 unselected subjects, referred for bone density, precision (CV%) measured on 2 occasions, separated by repositioning, ranged from 1.7% (mid-femur) to 4.5% at the lowest leg site. Intra-operator precision, measured by three operators on ten subjects on three occasions, was between 1.0% and 2.9%, whilst inter-operator precision was between 1.0% and 3.6%, according to region. These values compare well with those at the spine and upper femur, and in the literature. There was no evidence that this operator agreement improved between occasions 1 and 3. This technique promises to be useful for assessing bone changes at vulnerable sites in the lower limb, in diverse pathological states and in assessing response to treatment.

High precision semiautomated computed tomography measurement of lumbar disk and vertebral heights

Evaluation of treatments of many spine disorders requires precise measurement of the heights of vertebral bodies and disk spaces. The authors present a semiautomated computer algorithm measuring those heights from spine computed tomography (CT) scans and evaluate its precision. Eight patients underwent two spine CT scans in the same day. In each scan, five thoracolumbar vertebral heights and four disk heights were estimated using the algorithm. To assess precision, the authors computed the differences between the height measurements in the two scans, coefficients of variation (CV), and 95% limits of agreement. Intraoperator and interoperator precisions were evaluated. For local vertebral and disk height measurement (anterior, middle, posterior) the algorithm was compared to a manual mid-sagittal plane method. The mean (standard deviation) interscan difference was as low as 0.043 (0.031) mm for disk heights and 0.044 (0.043) mm for vertebral heights. The corresponding 95% limits of agreement were [-0.085, 0.11] and [-0.10, 0.12] mm, respectively. Intraoperator and interoperator precision was high, with a maximal CV of 0.30%. For local vertebral and disk heights, the algorithm improved upon the precision of the manual mid-sagittal plane measurement by as much as a factor of 6 and 4, respectively. The authors evaluated the precision of a novel computer algorithm for measuring vertebral body heights and disk heights using short term repeat CT scans of patients. The 95% limits of agreement indicate that the algorithm can detect small height changes of the order of 0.1 mm.

Automated Segmentation of the Lumbar Pedicle in CT Images for Spinal Fusion Surgery

Exact information about the shape of a lumbar pedicle can increase operation accuracy and safety during computer-aided spinal fusion surgery, which requires extreme caution on the part of the surgeon, due to the complexity and delicacy of the procedure. In this paper, a robust framework for segmenting the lumbar pedicle in computed tomography (CT) images is presented. The framework that has been designed takes a CT image, which includes the lumbar pedicle as input, and provides the segmented lumbar pedicle in the form of 3-D voxel sets. This multistep approach begins with 2-D dynamic thresholding using local optimal thresholds, followed by procedures to recover the spine geometry in a high curvature environment. A subsequent canal reference determination using proposed thinning-based integrated cost is then performed. Based on the obtained segmented vertebra and canal reference, the edge of the spinal pedicle is segmented. This framework has been tested on 84 lumbar vertebrae of 19 patients requiring spinal fusion. It was successfully applied, resulting in an average success rate of 93.22 % and a final mean error of 0.14 ± 0.05 mm. Precision errors were smaller than 1 % for spine pedicle volumes. Intra- and interoperator precision errors were not significantly different.