Laboratory Workflow Research Articles

Proteogenomic detection of circulating biomarkers for clinical oncology.

e15010 Background: Circulating biomarkers have the potential to detect cancer in its earliest stages and monitor patients in remission. The integration of proteogenomics in circulating biomarkers may transform the molecular diagnostics of cancer and accelerate basic and clinical oncology research. Proteomics bridges the gaps of functional information lost due to post-transcriptional and post-translational modifications in a genomic approach. A recent study showed that adding just 8 protein biomarkers to a panel of circulating DNA biomarkers increased the diagnostic accuracy up to 98% sensitivity and 99% specificity. However, the proteogenomic approach normally requires the use of multiple different assay technologies and laboratory workflows, including mass spectrometry. Methods: MosaicNeedles are densely integrated nanoneedle sensors fabricated on a planar substrate that integrates proteogenomic analysis in one platform. 94,000 sensors with more than 2 billion total nanoneedles can be integrated on to a standard SBS plate, which can be configured into 96, 384 or 1536 well format. Each sensor contains an array of nanoneedles, dedicated to detecting one analyte of interest. All nanoneedles comprising the same sensor are functionalized with the same capture probes. The capture probe can be either an antibody for protein detection or an oligonucleotide with a specific target sequence to a DNA fragment, mRNA, or miRNA of interest. Results: At low analyte concentration, the binding of proteins to the nanoneedles follows a Poisson distribution. Therefore, statistically, no more than one molecule is bound per nanoneedle. A further addition of aptamers or antibodies will form a sandwich complex with the target analyte. Since each of the nanoneedles has an intrinsic optical resonance spectrum and will red-shift as the sandwich complex forms on its surface, the number of analytes can be quantitated by simply counting the number of nanoneedles that display a color change. At high analyte concentration, each nanoneedle has more than one analyte, so the number of analytes can be calculated by averaging the spectrum shifts of all nanoneedles. This combined single molecule counting (digital) and spectrum shift (analog) analysis allows the platform to detect both high abundance and low abundance protein analytes in one reaction. A 10,000-plex study can be achieved with a total of 2.5 billion nanoneedles on a 50mm by 50mm consumable. In this consumable, a 2,000-plex proteome and 8,000 cell-free DNA fragments can be detected. Conclusions: A full proteogenomic quantification can be performed on the NanoMosaic platform in one reaction with high sensitivity and large dynamic range. It simplifies the workflow and allows users to integrate proteomic and genomic information to discover new circulating biomarkers.

7. Optical genome mapping for prenatal diagnostic testing

The current standard-of-care (SOC) for diagnostic prenatal testing incorporates a combination of cytogenetic technologies that include karyotyping, FISH, and chromosomal microarray (CMA) using cultured amniocytes or chorionic villi sampling (CVS). However, each technology has certain limitations viz. karyotyping has low resolution (>5Mb), FISH is targeted, and CMA does not detect balanced SVs or decipher complex rearrangements in the genome. The result of these limitations is the requirement to use multiple technologies to reach a genetic diagnosis. The current diagnostic prenatal testing workflow demonstrates the need for an alternative technology that can provide results in a cost and time-effective manner. Optical genome mapping (OGM) is an emerging technology that has demonstrated its ability to detect all classes of structural variants (SVs), including copy number variations (CNVs) and balanced aberrations in a single assay, but has not been evaluated in prenatal setting. Herein, we evaluated 80 prenatal amniocyte cases using OGM that were previously characterized by SOC technologies (Karyotype and FISH/CMA). Three samples were run in triplicate to evaluate inter-run, intra-run and inter-instrument reproducibility. Clinically relevant SVs and CNVs were reported using built-in filtration criteria and phenotype-specific analysis. OGM data was found to be 100% concordant with conventional cytogenetic analyses. In addition to identifying previously characterized SVs, OGM identified additional aberrations and demonstrated a higher resolution and sensitivity to accurately define SVs w.r.t size and location. Furthermore, OGM demonstrated 100% technical and analytical reproducibility across runs and instruments. This study demonstrated that OGM has the potential to identify all classes of SVs, including complex structural rearrangements. OGM has a standardized laboratory workflow and reporting solution that can be easily adopted in routine clinical laboratories. In conclusion, OGM has the potential to replace the current SOC methods for prenatal diagnostic testing and can be used as first-tier genetic test in prenatal setting.

Script-based automation of analytical instrument software tasks.

In laboratories with multiple identical analytical instruments and consistent sample workflows, analysts frequently perform repetitive software control steps, yet automation options in vendor-supplied instrument software are generally limited and may not support the desired laboratory workflow. Scripts that automate tasks to monitor systems, streamline worklist creation, or minimize downtime can save valuable personnel time and reduce errors. AutoHotkey is a free, open-source scripting language for Windows that allows users with no programming experience to easily create scripts automating a wide variety of activities. The scripts automate the tasks that a user performs while interacting with the instrument control software, such as mouse clicks and keyboard entries and closing software windows, rather than modify the underlying instrument software, and thus these scripts are compatible with multiple vendor software packages and Windows OS versions. The scripts can be triggered manually from a desktop icon or automatically through Windows Task Scheduler.

32. Genetic testing for individuals with non-syndromic differences in sex development (DSD): collaborative approach yields results

Differences of Sex Development (DSD) are a group of conditions in which there is atypical anatomic, gonadal, or chromosomal sex. With input from multiple stakeholders, our laboratory recently validated a genetic testing panel for non-syndromic DSD, which utilizes targeted next generation sequencing and exon-level microarray analysis. Data from the first 37 consecutive tests is presented here. The majority of patients in this cohort were evaluated in our institution's DSD clinic, which includes providers specializing in pediatric and reproductive endocrinology, genetics, urology, gynecology and child and adolescent psychology. Almost all patients had prior karyotype, SRY FISH, and/or chromosomal microarray testing. Exclusion criteria for panel testing included the presence of syndromic features or suspected 21-hydryoxylase deficiency. Pathogenic or likely pathogenic variants in genes associated with dominant conditions were reported for 15 (40.5%) individuals. The pathogenic and likely pathogenic variants were identified in 10 different genes: AMH, AMHR2, ANOS1, AR (in 5 unrelated individuals), CYP17A1, MAMLD1 (in two unrelated individuals), MAP3K1, PROKR2, SRY, and STAR. For 3 other individuals (8%), a single pathogenic variant was reported in a gene associated with a recessive condition. Variants of uncertain clinical significance were reported for 5 individuals (13.5%), and no variants were reported for 14 individuals (38%). Close collaboration between the laboratory and the clinical team during the test validation process, as well as ongoing discussions surrounding test utilization, enabled development of a panel most appropriate for our patient population, with a high diagnostic rate. The targeted panel approach avoided issues related to incidental findings and contributed to the lower rate of reported variants of uncertain significance. Future goals for the DSD panel include streamlining the laboratory workflow and regular updates to panel content to include genes recently implicated in DSD-related conditions.

Reducing Laboratory Turnaround Time in Patients With Acute Stroke and the Lack of Impact on Time to Reperfusion Therapy.

Timely reperfusion improves the recovery of patients with acute ischemic stroke. Laboratory results are crucial to guide treatment decisions in patients when abnormal laboratory tests are suspected. To implement a new laboratory workflow for acute stroke patients and compare laboratory turnaround time (TAT) preimplementation and postimplementation. We conducted a retrospective pre-post intervention study of patients with suspected acute stroke during the 4-month periods before and after the implementation of a new laboratory workflow process. The improvement process included relocating the specimen registration site, laboratory notification before specimen arrival, a color-coding system on tubes, timing at all processes, and eliminating the smear review if platelets were normal. TATs of the laboratory and door-to-clinical intervention times before and after the improvement process were compared. Postintervention, median specimen transportation time decreased from 11 (interquartile range [IQR], 8.4-16.4) to 9 minutes (IQR, 6.3-12.8), P < .001. The intralaboratory and total TATs of complete blood cell count, coagulation tests, and creatinine significantly decreased (P < .001 for all). Blood drawn-to-laboratory reported time decreased from 43 (IQR, 36.0-51.5) to 33 minutes (IQR, 29.2-35.8, P < .001). However, door-to-needle time for thrombolysis and door-to-puncture time and door-to-recanalization time for mechanical thrombectomy were not statistically different (P = .11, .69, and .50, respectively). The new laboratory workflow significantly decreased transportation time, TAT of individual tests, and the blood drawn-to-laboratory reported time. However, the time to treatment of acute ischemic stroke patients was not different between preimplementation and postimplementation.

Clinical implementation of RNA sequencing for Mendelian disease diagnostics

BackgroundLack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics.MethodsWe implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage.ResultsWe detected on average 12,500 genes per sample including around 60% of all disease genes—a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions.ConclusionTogether, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

An Update on Laboratory-Based Diagnostic Biomarkers for Multiple Sclerosis and Beyond.

Multiple sclerosis (MS) is an immune-mediated central nervous system (CNS) inflammatory demyelinating disease in which analysis of clinical presentation, imaging studies, and laboratory tests aid in diagnosis. This review discusses laboratory tests ordered to rule out and rule in MS, such as the traditional measurement of cerebrospinal fluid (CSF) IgG index and oligoclonal bands. Biomarkers discovered in the past 2 decades, such as aquaporin-4 (AQP4) antibodies and myelin oligodendrocyte glycoprotein (MOG) antibodies, have been incorporated into clinical practice in the diagnosis of disorders referred to as MS mimics. The importance of test selection, assay methodology, optimal sample for testing, and diagnostic utility of these biomarkers is reviewed. Other laboratory testing that can aid in the differentiation between MS and these biomarker-defined CNS demyelinating diseases is described. There is a focus on emerging biomarkers such as the use of kappa immunoglobulin free light chain concentration in CSF and kappa CSF index measurement as an alternative to oligoclonal bands which has a potential for an improvement in laboratory workflows. Finally, the role of biomarkers of disease activity and prognosis are discussed, including neurofilament light chain, glial fibrillary acidic protein, and myelin basic protein. Future perspectives with improved laboratory testing tools and discovery of additional biomarkers are provided. Laboratory testing for demyelinating disorders using CSF and serum are routine practices that can benefit from an update, as novel biomarker-defined entities have reduced the potential for MS misdiagnosis, and CSF/serum biomarkers reinstated in the diagnostic criteria of MS.

The application of next generation and whole genome sequencing in the diagnosis of hematological disorders and challenges to apply in routine diagnosis

By assessing a patient's genetic makeup at a low cost, next-generation sequencing (NGS) technologies have begun to change the area of hematological malignancies. High-throughput, massively parallel DNA sequencing technologies are rapidly revealing new information in hematology, cancer, clinical genetics, and a variety of other diseases. It provides researchers with a new viewpoint on the onset of sickness, risk assessment, and therapeutic action. In hematology these technologies are used to diagnose hematological malignancies, inherited coagulation bleeding disorders, minimal residual diseases, hereditary hemolytic anemia, and blood typing. The use of next-generation sequencing in regular diagnostic procedures raises a number of challenges, including result interpretation, laboratory workflow, data storage, and ethical concerns.

Maintaining laboratory quality assurance and safety in a pandemic: Experiences from the KEMRI-Wellcome Trust Research Programme laboratory’s COVID-19 response

Laboratory diagnosis plays a critical role in the containment of a pandemic. Strong laboratory quality management systems (QMS) are essential for laboratory diagnostic services. However, low laboratory capacities in resource-limited countries has made the maintenance of laboratory quality assurance, especially during a pandemic, a daunting task. In this paper, we describe our experience of how we went about providing diagnostic testing services for SARS-CoV-2 through laboratory reorganization, redefining of the laboratory workflow, and training and development of COVID-19 documented procedures, all while maintaining the quality assurance processes during the COVID-19 pandemic at the Kenya Medical Research Institute (KEMRI) Wellcome Trust Research Programme (KWTRP) laboratory. The KWTRP laboratory managed to respond to the COVID-19 outbreak in Kenya by providing diagnostic testing for the coastal region of the country, while maintaining its research standard quality assurance processes. A COVID-19 team comprising of seven sub-teams with assigned specific responsibilities and an organizational chart with established reporting lines were developed. Additionally, a total of four training sessions were conducted for county Rapid Response Teams (RRTs) and laboratory personnel. A total of 11 documented procedures were developed to support the COVID-19 testing processes, with three for the pre-analytical phases, seven for the analytical phase, and one for the post-analytical phase. With the workflow re-organization, the development of appropriate standard operating procedures, and training, research laboratories can effectively respond to pandemic outbreaks while maintaining research standard QMS procedures.

Clinical validation of a multiplex PCR-based detection assay using saliva or nasopharyngeal samples for SARS-Cov-2, influenza A and B

The COVID-19 pandemic has resulted in significant diversion of human and material resources to COVID-19 diagnostics, to the extent that influenza viruses and co-infection in COVID-19 patients remains undocumented and pose serious public-health consequences. We optimized and validated a highly sensitive RT-PCR based multiplex-assay for the detection of SARS-CoV-2, influenza A and B viruses in a single-test. This study evaluated clinical specimens (n = 1411), 1019 saliva and 392 nasopharyngeal swab (NPS), tested using two-assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp-RT-PCR based assay that targets SARS-CoV-2, influenza viruses A and B. Of the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. This study presents clinical validation of a multiplex-PCR assay for testing SARS-CoV-2, influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow.

Machine learning for detecting wrong blood in tube errors

Wrong blood in tube (WBIT) errors are a persisting patient safety issue in clinical laboratory practice. Post-analytically, these errors are identified by a combination of automated delta checks and manual review of results by laboratory staff. However, machine learning may offer a superior approach. Evidence is emerging that machine learning models outperform both delta checks and review of results by laboratory staff. This finding challenges the assumption of human superiority on the task and highlights the need to establish new laboratory workflows that optimise contributions from both machine learning and staff.

357 A Novel Protocol for Post-Operative Bloods Following Elective Colorectal Cancer Resections - a Re-Audit of a Closed Loop

Abstract Aim Indications for blood tests are often poorly considered. Appropriate blood requests reduce trauma for patients, improve laboratory workflow and reduce costs. We therefore created a novel protocol for bloods requested in the post-operative period for patients undergoing elective colorectal cancer resections. We performed two audit cycle loops to assess protocol adherence and evaluate financial savings. Method The protocol was agreed locally by consultant consensus, presented locally in February 2021, and displayed on surgical wards in May 2021. Bloods requested were reviewed retrospectively for patients undergoing elective colonic resection with an uncomplicated post-operative course. Data was collected prior to the local meeting between September-November 2020 (audit 1, cycle 1), after the meeting between March-April 2021 (audit 2, cycle 1), then after distribution of the protocol to wards between July-August 2021 (cycle 2). Results The locally agreed protocol was post-op day 1,5,7 FBC, U&amp;E, CRP; day 3 FBC, U&amp;E, CRP, LFT; day 2,4,6 no bloods. In audit 1 (cycle 1) 100% protocol adherence would have saved £15.96 per patient (n = 20). In audit 2 (cycle 1) the protocol was followed on 21.4% of days with further savings of £14.21 per patient (n = 23) had there been 100% adherence. In cycle 2, there was 40.74% protocol adherence with £6.62 further potential savings per patient (n = 15). Conclusions Following the post-elective colorectal resection bloods protocol may save our hospital £1700 per annum. Protocol adherence improved when displayed on surgical wards. With these exciting results, we are working with Medway EPR to create an order set containing this protocol.

Trio genome sequencing for developmental delay and pediatric heart conditions: A comparative microcost analysis

PurposeGenome sequencing (GS) can aid clinical management of multiple pediatric conditions. Insurers require accurate cost information to inform funding and implementation decisions. The objective was to compare the laboratory workflows and microcosts of trio GS testing in children with developmental delay (DD) and in children with cardiac conditions. MethodsCost items related to each step in trio GS (child and 2 parents) for both populations were identified and measured. Program costs over 5 years were estimated. Probabilistic and deterministic analyses were conducted. ResultsThe mean cost per trio GS was CAD$6634.11 (95% CI = 6352.29-6913.40) for DD and CAD$8053.10 (95% CI = 7699.30-8558.10) for cardiac conditions. The 5-year program cost was CAD$28.11 million (95% CI = 26.91-29.29) for DD and CAD$5.63 million (95% CI = 5.38-5.98) for cardiac conditions. Supplies constituted the largest cost component for both populations. The higher cost per sample for the population with cardiac conditions was due to the inclusion of pharmacogenomics, higher bioinformatics labor costs, and a more labor intensive case review. ConclusionThis analysis indicated important variation in trio GS workflow and costs between pediatric populations in a single institution. Enhanced understanding of the clinical utility and costs of GS can inform harmonization and implementation decision-making.

Is faster better? An economic evaluation of rapid and ultra-rapid genomic testing in critically ill infants and children

PurposeTo evaluate whether the additional cost of providing increasingly faster genomic results in pediatric critical care is outweighed by reductions in health care costs and increases in personal utility. MethodsHospital costs and medical files from a cohort of 40 children were analyzed. The health economic impact of rapid and ultra-rapid genomic testing, with and without early initiation, relative to standard genomic testing was evaluated. ResultsShortening the time to results led to substantial economic and personal benefits. Early initiation of ultra-rapid genomic testing was the most cost-beneficial strategy, leading to a cost saving of AU$26,600 per child tested relative to standard genomic testing and a welfare gain of AU$12,000 per child tested. Implementation of early ultra-rapid testing of critically ill children is expected to lead to an annual cost saving of AU$7.3 million for the Australian health system and an aggregate welfare gain of AU$3.3 million, corresponding to a total net benefit of AU$10.6 million. ConclusionEarly initiation of ultra-rapid genomic testing can offer substantial economic and personal benefits. Future implementation of rapid genomic testing programs should focus not only on optimizing the laboratory workflow to achieve a fast turnaround time but also on changing clinical practice to expedite test initiation.

Development and validation of a high throughput SARS-CoV-2 whole genome sequencing workflow in a clinical laboratory

Monitoring new mutations in SARS-CoV-2 provides crucial information for identifying diagnostic and therapeutic targets and important insights to achieve a more effective COVID-19 control strategy. Next generation sequencing (NGS) technologies have been widely used for whole genome sequencing (WGS) of SARS-CoV-2. While various NGS methods have been reported, one chief limitation has been the complexity of the workflow, limiting the scalability. Here, we overcome this limitation by designing a laboratory workflow optimized for high-throughput studies. The workflow utilizes modified ARTIC network v3 primers for SARS-CoV-2 whole genome amplification. NGS libraries were prepared by a 2-step PCR method, similar to a previously reported tailed PCR method, with further optimizations to improve amplicon balance, to minimize amplicon dropout for viral genomes harboring primer-binding site mutation(s), and to integrate robotic liquid handlers. Validation studies demonstrated that the optimized workflow can process up to 2688 samples in a single sequencing run without compromising sensitivity and accuracy and with fewer amplicon dropout events compared to the standard ARTIC protocol. We additionally report results for over 65,000 SARS-CoV-2 whole genome sequences from clinical specimens collected in the United States between January and September of 2021, as part of an ongoing national genomics surveillance effort.

Total Laboratory Automation for Rapid Detection and Identification of Microorganisms and Their Antimicrobial Resistance Profiles.

At a time when diagnostic bacteriological testing procedures have become more complex and their associated costs are steadily increasing, the expected benefits of Total laboratory automation (TLA) cannot just be a simple transposition of the traditional manual procedures used to process clinical specimens. In contrast, automation should drive a fundamental change in the laboratory workflow and prompt users to reconsider all the approaches currently used in the diagnostic work-up including the accurate identification of pathogens and the antimicrobial susceptibility testing methods. This review describes the impact of TLA in the laboratory efficiency improvement, as well as a new fully automated solution for AST by disk diffusion testing, and summarizes the evidence that implementing these methods can impact clinical outcomes.

Recovery Observations from Alkali, Nanoparticles and Polymer Flooding as Combined Processes.

We have studied wettability alterations through imbibition/flooding and their synergy with interfacial tension (IFT) for alkalis, nanoparticles and polymers. Thus, the total acid number (TAN) of oil may determine the wetting-state of the reservoir and influence recovery and IFT. Data obtained demonstrate how the oil TAN number (low and high), chemical agent and reservoir mineralogy influence fluid–fluid and rock–fluid interactions. We used a laboratory evaluation workflow that combines complementary assessments such as spontaneous imbibition tests, IFT, contact angle measurements and selected core floods. The workflow evaluates wettability alteration, IFT changes and recovery when injecting alkalis, nanoparticles and polymers, or a combination of them. Dynamics and mechanisms of imbibition were tracked by analyzing the recovery change with the inverse bond number. Three sandstone types (outcrops) were used, which mainly differed in clay content and permeability. Oils with low and high TANs were used, the latter from the potential field pilot 16 TH reservoir in the Matzen field (Austria). We have investigated and identified some of the conditions leading to increases in recovery rates as well as ultimate recovery by the imbibition of alkali, nanoparticle and polymer aqueous phases. This study presents novel data on the synergy of IFT, contact angle Amott imbibition, and core floods for the chemical processes studied.

A Sensitive and Quantitative Assay to Enumerate and Measure Mycobacterium leprae Viability in Clinical and Experimental Specimens.

Mycobacterium leprae, the etiologic agent of leprosy, cannot be cultured on artificial media. This characteristic, coupled with its long generation time, presents a number of unique challenges to studying this pathogen. One of the difficulties facing both researchers and clinicians is the absence of a rapid test to measure the viability of M. leprae in clinical or experimental specimens. The lack of such a tool limits the understanding of M. leprae immunopathogenesis and makes determining the efficacy of drug treatments difficult. With this in mind, we developed a robust two-step molecular viability assay (MVA) that first enumerates the M. leprae in the tissue; then, this data is used to normalize bacterial RNA quantities for the second step, in which the expression of M. leprae esxA and hsp18 are measured. This assay is specific and sensitive enough to be used on most clinical samples. This protocol describes the steps required to extract DNA and RNA from M. leprae-infected tissue, enumerate M. leprae, and measure M. leprae viability based on the normalized expression of two M. leprae-specific genes (hsp18 and esxA). This protocol also outlines an optimal laboratory design and workflow for performing this assay. © 2022 The Leprosy Mission Nepal. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: DNA and RNA P purification from M. leprae-infected tissue Basic Protocol 2: Enumeration of M. leprae by RLEP qPCR on the DNA fraction Basic Protocol 3: Calculation of M. leprae per tissue and normalization of RNA Basic Protocol 4: Reverse-transcription of normalized RNA to generate cDNA Basic Protocol 5: Determination of M. leprae viability using HSP18 and ESXA qPCR on the cDNA Support Protocol 1: M. leprae qPCR primer/probe stock preparation Support Protocol 2: Preparation of plasmid stocks and standard curves.

Three-dimensional printing of clinical scale and personalized calcium phosphate scaffolds for alveolar bone reconstruction

ObjectiveAlveolar bone defects can be highly variable in their morphology and, as the defect size increases, they become more challenging to treat with currently available therapeutics and biomaterials. This investigation sought to devise a protocol for fabricating customized clinical scale and patient-specific, bioceramic scaffolds for reconstruction of large alveolar bone defects. MethodsTwo types of calcium phosphate (CaP)-based bioceramic scaffolds (alginate/β-TCP and hydroxyapatite/α-TCP, hereafter referred to as hybrid CaP and Osteoink™, respectively) were designed, 3D printed, and their biocompatibility with alveolar bone marrow stem cells and mechanical properties were determined. Following scaffold optimization, a workflow was developed to use cone beam computed tomographic (CBCT) imaging to design and 3D print, defect-specific bioceramic scaffolds for clinical-scale bone defects. ResultsOsteoink™ scaffolds had the highest compressive strength when compared to hybrid CaP with different infill orientation. In cell culture medium, hybrid CaP degradation resulted in decreased pH (6.3) and toxicity to stem cells; however, OsteoInk™ scaffolds maintained a stable pH (7.2) in culture and passed the ISO standard for cytotoxicity. Finally, a clinically feasible laboratory workflow was developed and evaluated using CBCT imaging to engineer customized and defect-specific CaP scaffolds using OsteoInk™. It was determined that printed scaffolds had a high degree of accuracy to fit the respective clinical defects for which they were designed (0.27 mm morphological deviation of printed scaffolds from digital design). SignificanceFrom patient to patient, large alveolar bone defects are difficult to treat due to high variability in their complex morphologies and architecture. Our findings shows that Osteoink™ is a biocompatible material for 3D printing of clinically acceptable, patient-specific scaffolds with precision-fit for use in alveolar bone reconstructive procedures. Collectively, emerging digital technologies including CBCT imaging, 3D surgical planning, and (bio)printing can be integrated to address this unmet clinical challenge.

Analysis of mixed DNA profiles from the RapidHIT™ ID platform using probabilistic genotyping software STRmix™

Rapid DNA instruments are gaining interest in the forensic community as a means to generate DNA profile information more quickly than standard laboratory workflows, and with the potential to be carried out at the scene where samples are taken. Due to the many years that DNA profiles have been generated in a standard laboratory workflow, there have been numerous studies into profiling performance. These have flowed into probabilistic means of evaluating DNA profiles produced in those laboratories. In this study we examined the DNA profiling performance of the RapidHIT™ ID system on artificial mixtures constructed from raw DNA, mixtures constructed from body fluids, and touch DNA samples. We calibrated the probabilistic genotyping system STRmix™ for use on results produced by the RapidHIT ID system. Identical DNA samples were split, with half analysed on the RapidHIT ID system and the other half analysed in the standard laboratory workflow. Profiles produced from these paired samples were compared with regards to their composition and discrimination power. In general, profiles produced using the RapidHIT ID system showed good discrimination power, but less than those produced via the standard laboratory workflow. This is the expected trade-off for the advantages of speed and portability.