Small Clinical Laboratories Research Articles

LBA02-10 ACCURACY OF A SIMPLIFIED SEQUENCING AND BIOINFORMATICS PLATFORM ON GENETIC VARIANT DETECTION IN PROSTATE CANCER PATIENTS

You have accessJournal of UrologyCME1 Apr 2023LBA02-10 ACCURACY OF A SIMPLIFIED SEQUENCING AND BIOINFORMATICS PLATFORM ON GENETIC VARIANT DETECTION IN PROSTATE CANCER PATIENTS Deepak Kapoor, Jonathan Anderson, and Ann Anderson Deepak KapoorDeepak Kapoor More articles by this author , Jonathan AndersonJonathan Anderson More articles by this author , and Ann AndersonAnn Anderson More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003361.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Consensus guidelines suggest that genetic testing is clinically indicated for prostate cancer (PCa) patients who meet certain family history, stage and histopathological grade criteria. Current next generation sequencing (NGS) diagnostics rely on capital and maintenance intensive sequencers and require highly trained technical personnel to operate. This may restrict availability of testing where specimen transport or access to personnel/capital is limited. We sought to compare a simpler sequencer coupled with a bioinformatics platform which requires minimal maintenance and capital cost with traditional NGS equipment. METHODS: We compared results in detection of 10 genes recommended by NCCN PCa guidelines (ATM, BRCA1, BRCA2, CHEK2, MLH1, MSH2, MSH6, PALB2, PSM2, and HOXB13) between the Oxford Nanopore Technologies MinION to the Illumina NextSeq sequencer. All samples were sequenced in a depth greater than 50x. Bioinformatics for the Illumina sequencer was Illumina’s TruSight™ Hereditary Cancer panel. As the MinION reads long DNA fragments, and cancer diagnostics is focused on short exon sequencing, we used the CorrSeq Dx™ by Foresee Genomics to create long DNA molecules consisting of several tandem repeats of short DNA fragments separated by sequences recognized by the automated bioinformatics software. Statistical analysis was performed using Cohen’s kappa test. This IRB-exempt study was conducted in accordance with the Declaration of Helsinki. RESULTS: We detected using 380 variants in 13 patients on the Illumina platform and 379 (3 false negatives, 2 false positives) variants on the same patients using the MinION sequencer with CorrSeq Dx assay. The positive predictive value and sensitivity for the simplified platform was 99.5% (375/(375+2)) and 99.2% (375/(375+3)), respectively. The demonstrated accuracy of the simplified process was 99.9% (Cohen's kappa >0.99). CONCLUSIONS: Our data suggests that this application could allow smaller clinical laboratories which are currently outsourcing their NGS diagnostics to perform genetic testing in-house. Use of a simpler process for NGS testing could improve access to guideline-based care in remote and economically disadvantaged areas, resulting in better treatment outcomes as well as increased global healthcare equity. Further testing of this technology is warranted to further assess its impact on patient care. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1190 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Deepak Kapoor More articles by this author Jonathan Anderson More articles by this author Ann Anderson More articles by this author Expand All Advertisement PDF downloadLoading ...

Development of a Fully Automated Desktop Analyzer and Ultrahigh Sensitivity Digital Immunoassay for SARS-CoV-2 Nucleocapsid Antigen Detection.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. Methods: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. Results: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. Conclusions: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.

SCHOOL: Software for Clinical Health in Oncology for Omics Laboratories

Bioinformatics analysis is a key element in the development of in-house next-generation sequencing assays for tumor genetic profiling that can include both tumor DNA and RNA with comparisons to matched-normal DNA in select cases. Bioinformatics analysis encompasses a computationally heavy component that requires a high-performance computing component and an assay-dependent quality assessment, aggregation, and data cleaning component. Although there are free, open-source solutions and fee-for-use commercial services for the computationally heavy component, these solutions and services can lack the options commonly utilized in increasingly complex genomic assays. Additionally, the cost to purchase commercial solutions or implement and maintain open-source solutions can be out of reach for many small clinical laboratories. Here, we present Software for Clinical Health in Oncology for Omics Laboratories (SCHOOL), a collection of genomics analysis workflows that (i) can be easily installed on any platform; (ii) run on the cloud with a user-friendly interface; and (iii) include the detection of single nucleotide variants, insertions/deletions, copy number variants (CNVs), and translocations from RNA and DNA sequencing. These workflows contain elements for customization based on target panel and assay design, including somatic mutational analysis with a matched-normal, microsatellite stability analysis, and CNV analysis with a single nucleotide polymorphism backbone. All of the features of SCHOOL have been designed to run on any computer system, where software dependencies have been containerized. SCHOOL has been built into apps with workflows that can be run on a cloud platform such as DNANexus using their point-and-click graphical interface, which could be automated for high-throughput laboratories.

Comparison of analytical performance of the HbA1c assay on the fine care fluorescence immunochromatographic system.

Background: The Fluorescence Immunochromatographic method is a relatively new technique offering similar advantages to small clinical laboratories with the added value of reagents stability and increased precision. Here we aim to evaluate the Fluorescence Immunochromatographic method in terms of accuracy, precision, and linearity. Also, to compare its performance against the Bronate Affinity method. Methods: Reproducibility between the two methods was assessed by measuring ten prepared HbA1c reference materials with different concentrations. Additionally, the Bland-Altman Plot was also used to determine the comparability of measurements between the two methods within a 1.96 SD limit. Moreover, twenty repeated measurements of two HbA1c reference materials with different concentrations were carried out to assess accuracy and precision. Results: The “best fit values” after comparing ten measurements of the HbA1c reference materials between the two methods for the Slope of the linear regression model was 0.9764 ± 0.05566 with “Y” intercept of 0.5469 ± 0.4657, “X” intercept of -0.5601, and R2 of 0.9747. All ten results were inside the 1.96 limit of the Bland-Altman Plot. Similarly, comparison of two sets of HbA1c references materials, “Control N” and “Control P”, after twenty repeated analysis showed a Coefficient of Variation (CV) of 2.64% and 3.55% respectively. Conclusion: There is no significant difference between the two methods in detecting HbA1c. Method evaluation employing both a liner regression model of analysis as well as the Bland-Altman method showed no significant variance. This shows that the Fluorescent Immunochromatography instrument is suitable for detecting HbA1c in the clinically significant detection range.

WarmStart colorimetric RT-LAMP for the rapid, sensitive and specific detection of Enteroviruses A-D targeting the 5'UTR region.

The aim of the present study was to develop a colorimetric LAMP assay for the detection of enteroviruses belonging to species A-D targeting the 5' untranslated region (5' UTR) of enteroviruses genome. The RNA was converted to cDNA by the reverse transcriptase and then amplified via LAMP by the WarmStart®Bst DNA polymerase, simultaneously in a single reaction tube, so we shortened the reaction time to 50min. The sensitivity of the assay regarding Enterovirus B, C and D was determined to be 0·30 CCID50 assay-1 while the sensitivity for Enterovirus A was 3·00 CCID50 assay-1 . The assay demonstrated high specificity and sensitivity for the detection of 45 reference strains of Enteroviruses A-D and validated on 20 clinical isolates. This assay can be used as a diagnostic tool for the rapid, sensitive and specific detection of enteroviruses, easily implemented in small clinical and research laboratories since LAMP amplicons were visualized by colour changes eliminating the requirement for post-amplification processing steps. We developed a colorimetric assay ideal for field situations for the detection of enteroviruses, by targeting the 5' UTR. This assay demonstrated high specificity and sensitivity, based on its performance on 45 EV A-D reference strains, on 20 EV B clinical isolates and on three non-enteroviral RNA viruses.

Is spectrophotometric enzymatic method a cost-effective alternative to indirect Ion Selective Electrode based method to measure electrolytes in small clinical laboratories?

Abstract Aim Our study was aimed to compare two different laboratory methods used to measure serum sodium and serum potassium level i.e. between the indirect Ion Selective Electrode (ISE) based and Enzymatic based method in Beckman Coulter AU-480, USA, in serum samples. Materials and methods This was a cross sectional single center study carried out from September’16 to January’17 on 130 serum samples, to compare the result of serum sodium (S. Na+) and serum potassium (S. K+) level by indirect ISE and Enzymatic method in fully automated biochemistry analyzer (Beckman Coulter AU-480, USA). Results To validate new enzymatic method bias, CV% and correlation coefficient were measured for S. Na+ and S. K+. The study has drawn a considerable agreement for measurement of S. K+ level between two methods with mean ± SD of difference was 0.14 ± 0.22 (p = 0.2242), CV% 2.7% and r2 value of 0.94, whereas disagreement for measurement of S. Na+ level with mean ± SD of difference was 2.38 ± 2.36 (p < 0.01), CV% 0.5% and r2 value of 0.82. Bias, CV% and correlation coefficient are acceptable for S. K+, whereas nonacceptable for S. Na+ level measurement as per Clinical Laboratory Improvement Amendments (CLIA) guidelines. Conclusion There was a good correlation in S. K+ between both methods whereas values were not correlated well with S. Na+ level. So, enzymatic method can be used as an alternative to indirect ISE method for estimation of S. K+ in small laboratories due to its cost effectiveness, but still intense workout is required for estimation of S. Na+.

WarmStart colorimetric LAMP for the specific and rapid detection of HPV16 and HPV18 DNA

Background and objectivesPersistent infection with High-Risk HPV genotypes is the principal cause for the development of cervical cancer with HPV16 and HPV18 to be the most frequently identified HPV genotypes observed in approximately 70% of cervical cancer cases worldwide. The present study focused on the development of a simple molecular methodology based on WarmStart colorimetric LAMP for the specific identification of HPV16 and HPV18. MethodsThe method was developed by designing LAMP type-specific primer sets that target the E6 gene. The assay was applied using HPV-positive clinical samples along with control cases in order to evaluate the specificity of the newly designed isothermal protocol. In addition, an experimental cutoff value was estimated through reconstitution experiments with HPV-DNA plasmids. LAMP amplicons were visualized by color changes, thus eliminating the requirement for post-amplification processing steps. ResultsThe WarmStart colorimetric LAMP facilitates the isothermal amplification of 10 copies per reaction of both HPV16 and HPV18 DNA, while it exhibits 100% specificity for the detection of the corresponding genotypes in LSIL and HSIL cases. Moreover, the assay demonstrates 100% PPV and 100% NPV. Finally, the sensitivity of conventional PCR with the type-specific LAMP primer sets (B3/F3) for the HPV16, HPV18 DNA detection was 100 copies/reaction and 10 copies/reaction, respectively. ConclusionsThe newly established WarmStart colorimetric LAMP can be considered as a powerful molecular tool that it can be easily implemented in small clinical and research laboratories for a rapid and efficient identification of the most tumorigenic HPV genotypes.

Determination of Calcium in Blood

Human blood is tested in clinical diagnostic laboratories for numerous indicators. One of these indicators is the concentration of calcium. This review presents a chronological development of procedures for determining the concentration of calcium in plasma, serum, and whole blood, which are based on various analytical methods such as titrimetry, manometry, X-ray fluorescence, flame photometry, spectrophotometry, spectrofluorimetry, potentiometry, flame atomic absorption, inductively coupled plasma atomic emission spectrometry, chromatography, and mass spectrometry. A brief overview of these methods and the results of comparative tests are given. Currently, potentiometric procedures for determining the concentration of calcium in plasma, serum, and whole blood are the most widespread in small, medium, and large clinical diagnostic laboratories. Flame photometric procedures often act as expert methods. Mass spectrometric procedures are used to validate reference sample solutions, while inductively coupled plasma atomic emission spectrometric procedures and chromatographic procedures are used in research organizations.

Determination of Sodium in Blood

Human blood is tested in clinical diagnostic laboratories for numerous indicators. One of these indicators is the concentration of sodium. The review presents a chronological development of methods for determining the concentration of sodium in plasma, serum, and whole blood, based on different analytical methods such as flame photometry, potentiometry, ion chromatography, spectrophotometry, gravimetry, and titrimetry. A brief overview of these methods and the results of comparative tests are given. Currently, potentiometric procedures for determining the concentration of sodium in plasma, serum, and whole blood are the most widespread in small, medium, and large clinical diagnostic laboratories. Flame photometric procedures often act as expert methods. Ion chromatography is mainly used in research organizations, while gravimetric and titrimetric procedures are rarely used at present.

Molecular Diagnostic Methods of Brucellosis: a Note on Pitfalls

Clinical manifestations of the brucellosis may show extensive appearance because of its wide signs and symptoms. Patients with brucellosis are usually symptomatically treated by using different antibiotics at private clinics due to misdiagnosis of clinical laboratory findings specially those chronic forms. These subjects will have been hospitalized with a complicated form of signs after changing a few therapies (1). At this time, classical methods such as culture based and serological procedures could not alone meet of clinicians’ demands. Many researchers have been being tried to introduce an alternative method better in accuracy, reliability and efficiency aspects. However, most reported comparison studies were not able to present desired results. There are not any significant points in existence of various and even contradictory results in laboratory interpretation reports. Those patients with recent brucellosis involvement or acute disease are better diagnosed than those hospitalized or chronic cases. Unfortunately, it is being observed the most researchers are not attending the patient clinical infectious form. Obviously, in-patients have more problematical conditions that results to have lower sensitivity rate in some test or even different from each other. Unfortunately, this concern has not been noticed in the majority of molecular epidemiology studies. Analyzed results of various tests are not fully in agreement with each other in these reports, confirming each test is better suited for some specific clinical conditions (2). Another important problem is applying home brew protocols in all comparative studies. This type of non-commercial diagnostic protocol are being surprisingly applied in most small Iranian clinical laboratories. These assays have been being followed from some reported papers without any proper optimization experiments or exact clinical trial specifically for Iranian populations, particularly when several of the effective parameters in the optimization may not be well reflected in these released reports. Some of them for proper optimization and standardization are mentioned in other studies (3). Proper selecting sampling is additional problems. Serum specimens would not be preferred samples since Brucella spp. agents are intracellular which is integrated in PMN cells. Serum specimens may give positive results, but in those has high antibody titer or acute cases. Although it is used as applied specimens instead of peripheral anti-coagulated blood (4). The chance of the recovery Brucella spp. DNA is obviously reduced in those chronic and complicated subjects. In addition, sampling from patients undergoing treatment may have low positive predictive value results (5,6). It would seem international approved extraction method accompanied with internal control as control the DNA isolation procedure and to check for possible PCR inhibition should be used in molecular diagnostic settings (4,7). Finally the last point, various pathogenic Brucella species involve in many countries. At present, we have no significant documentary reports in frequency rate of Brucella spp. in our community. Some reports represent considerable rate for B. abortus while some not. Therefore, applied protocol must be enough sensitive to detect and even differentiate at least B. abortus from B. melitensis, although some reports illustrate the presence of B. canis as well as in Iran (8).

Performance Verification of Alternative Quality Control Materials for Urine Albumin Assessment.

Accurate detection of urine albumin is important for evaluating the progression of diabetic kidney disease. However, two levels of daily quality control may not be practically feasible in some small clinical laboratories owing to a small number of patient samples and high costs. We aimed to prepare homemade quality control material (HQM) to measure urine albumin and then verify its performance. Normal saline solution and fresh mixed urine samples from five donors with serious kidney disease were used to prepare two levels of HQM (HQM1 and HQM2). Anhydrous ethylene glycol and sodium azide were used as antifreeze and as a preservative, respectively. Before being separated into Eppendorf tubes, 20 tests for HQM1 and HQM2 were performed, resulting in mean ± SD of 19.52 ± 0.91 mg/L and 105.28 ± 3.71 mg/L, respectively. After having been divided, the vial-to-vial variations of HQM1 and HQM2 were small (4.93% and 3.70%, respectively). The stability of HQM1 and HQM2 stored at 2 - 8°C was about 2 months and 80 days, respectively, and when stored at -20°C, remained stable for more than 8 months. After 1 - 8 months of cryopreservation at -20°C, once opened, the HQM in every Eppendorf tube could be kept for at least five days (CV < 6.1%). Our HQM stored at -20°C remained stable for a long time, and so could be considered as an alternative to standard QMs in the clinical laboratory.

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status.

Many neurodegenerative diseases arestill lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Oftenpotential new biomarkers do not make itinto the clinicdue to limitationsin their development andhigh costs.However,targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkersfor clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small moleculesin clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4). In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation. The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibratethe MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

Analysis of Metagenomics Next Generation Sequence Data for Fungal ITS Barcoding: Do You Need Advance Bioinformatics Experience?

During the last few decades, most of microbiology laboratories have become familiar in analyzing Sanger sequence data for ITS barcoding. However, with the availability of next-generation sequencing platforms in many centers, it has become important for medical mycologists to know how to make sense of the massive sequence data generated by these new sequencing technologies. In many reference laboratories, the analysis of such data is not a big deal, since suitable IT infrastructure and well-trained bioinformatics scientists are always available. However, in small research laboratories and clinical microbiology laboratories the availability of such resources are always lacking. In this report, simple and user-friendly bioinformatics work-flow is suggested for fast and reproducible ITS barcoding of fungi.

Determination of uric acid in blood

The review presents a chronological development of methods to determine the uric acid concentration in plasma, serum, and whole blood, based on various analytical methods such as spectrophotometry, spectrofluorimetry, chromatography, gas chromatography–mass spectrometry, amperometry, and calorimetry. A brief overview of these methods and the results of comparative tests are given. It was noted that enzymatic and nonenzymatic spectrophotometric procedures to determine the uric acid concentration in plasma, serum, and whole blood are currently, the most widespread in small, medium, and large clinical diagnostic laboratories. In addition, a number of enzymatic spectrophotometric methods and chromatographic methods are used as expert procedures. Chromatography–mass spectrometry procedures are often applied to certify standard uric acid samples in blood serum. Enzymatic and nonenzymatic spectrofluorimetric procedures are mostly used in scientific biomedical research. Amperometric procedures to determine the uric acid concentration in blood are rarely used in modern clinical and diagnostic practice, and some nonenzymatic spectrophotometric and calorimetric procedures are actually not used.

Development of cross-priming amplification assays for rapid and sensitive detection of Aeromonas hydrophila.

Aeromonas hydrophila has been increasingly implicated as the aetiologic agent of various human diseases. Therefore, reliable laboratory detection and identification of this bacterium has become clinically and epidemiologically desirable. We developed a nearly instrument-free, simple molecular method for rapid detection of Aer.hydrophila using a cross-priming amplification (CPA) assay with the desA gene as the target. The desA gene is crucial for the survival and growth of Aer.hydrophila under iron starvation. The results can be visualized as colour changes without opening the reaction tubes. No false-positive results were observed for the 33 non-Aer.hydrophila strains tested to evaluate assay specificity. The limit of detection for Aer.hydrophila was approximately 200copies of desA per reaction (on reference plasmids) and 5×10(3) CFUg(-1) Aer.hydrophila in simulated human stool, which is the same sensitivity as a qPCR assay. The performance of the CPA assay was also evaluated with 100 stool specimens from diarrhoea patients and 40 environmental water samples. In conclusion, the simplicity, cost-effectiveness and nearly instrument-free platform of the CPA assay make it practical for use in primary care facilities and smaller clinical laboratories. Aeromonas hydrophila is a human pathogen that infects via exposed wounds or ingestion of contaminated water and food. In this study, a CPA-based PCR method was developed for specific, rapid, cost-effective detection of Aer.hydrophila, and the test results could be visualized without opening the reaction tubes. This is the first report on the application of the CPA method for the detection of Aer.hydrophila. This novel method could be practical for use in primary care facilities and smaller clinical laboratories.

Cross-reactivity of steroid hormone immunoassays: clinical significance and two-dimensional molecular similarity prediction.

BackgroundImmunoassays are widely used in clinical laboratories for measurement of plasma/serum concentrations of steroid hormones such as cortisol and testosterone. Immunoassays can be performed on a variety of standard clinical chemistry analyzers, thus allowing even small clinical laboratories to do analysis on-site. One limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Interfering molecules include structurally related endogenous compounds and their metabolites as well as drugs such as anabolic steroids and synthetic glucocorticoids.MethodsCross-reactivity of a structurally diverse set of compounds were determined for the Roche Diagnostics Elecsys assays for cortisol, dehydroepiandrosterone (DHEA) sulfate, estradiol, progesterone, and testosterone. These data were compared and contrasted to package insert data and published cross-reactivity studies for other marketed steroid hormone immunoassays. Cross-reactivity was computationally predicted using the technique of two-dimensional molecular similarity.ResultsThe Roche Elecsys Cortisol and Testosterone II assays showed a wider range of cross-reactivity than the DHEA sulfate, Estradiol II, and Progesterone II assays. 6-Methylprednisolone and prednisolone showed high cross-reactivity for the cortisol assay, with high likelihood of clinically significant effect for patients administered these drugs. In addition, 21-deoxycortisol likely produces clinically relevant cross-reactivity for cortisol in patients with 21-hydroxylase deficiency, while 11-deoxycortisol may produce clinically relevant cross-reactivity in 11β-hydroxylase deficiency or following metyrapone challenge. Several anabolic steroids may produce clinically significant false positives on the testosterone assay, although interpretation is limited by sparse pharmacokinetic data for some of these drugs. Norethindrone therapy may impact immunoassay measurement of testosterone in women. Using two-dimensional similarity calculations, all compounds with high cross-reactivity also showed a high degree of similarity to the target molecule of the immunoassay.ConclusionsCompounds producing cross-reactivity in steroid hormone immunoassays generally have a high degree of structural similarity to the target hormone. Clinically significant interactions can occur with structurally similar drugs (e.g., prednisolone and cortisol immunoassays; methyltestosterone and testosterone immunoassays) or with endogenous compounds such as 21-deoxycortisol that can accumulate to very high concentrations in certain disease conditions. Simple similarity calculations can help triage compounds for future testing of assay cross-reactivity.

Bioinformatics pipelines for targeted resequencing and whole-exome sequencing of human and mouse genomes: a virtual appliance approach for instant deployment.

Targeted resequencing by massively parallel sequencing has become an effective and affordable way to survey small to large portions of the genome for genetic variation. Despite the rapid development in open source software for analysis of such data, the practical implementation of these tools through construction of sequencing analysis pipelines still remains a challenging and laborious activity, and a major hurdle for many small research and clinical laboratories. We developed TREVA (Targeted REsequencing Virtual Appliance), making pre-built pipelines immediately available as a virtual appliance. Based on virtual machine technologies, TREVA is a solution for rapid and efficient deployment of complex bioinformatics pipelines to laboratories of all sizes, enabling reproducible results. The analyses that are supported in TREVA include: somatic and germline single-nucleotide and insertion/deletion variant calling, copy number analysis, and cohort-based analyses such as pathway and significantly mutated genes analyses. TREVA is flexible and easy to use, and can be customised by Linux-based extensions if required. TREVA can also be deployed on the cloud (cloud computing), enabling instant access without investment overheads for additional hardware. TREVA is available at http://bioinformatics.petermac.org/treva/.

Use of Proficiency Testing as a Tool to Improve Quality in Microbiology Laboratories

Proficiency testing (PT) is a valuable tool for assessing laboratory performance and verifying the accuracy and reliability of test results. Participation is required by the Clinical Laboratory Improvement Amendments (CLIA) of 1988 for each of the microbiology subspecialties (bacteriology, mycobacteriology, mycology, parasitology, and virology), and the regulations include specific PT requirements for each subspecialty. To determine the use and perceived value of PT beyond meeting CLIA requirements, the Centers for Disease Control and Prevention funded a cooperative agreement with the Association of Public Health Laboratories to convene a series of focus groups to query laboratory professionals responsible for PT. The seven focus groups were comprised of 60 laboratory professionals representing large and small clinical laboratories, microbiology subspecialties, and public health. While participants acknowledged the need to perform PT to meet regulatory requirements, many also cited benefits and challenges beyond regulatory compliance.

Automated Digital Microfluidic Platform for Magnetic-Particle-Based Immunoassays with Optimization by Design of Experiments

We introduce an automated digital microfluidic (DMF) platform capable of performing immunoassays from sample to analysis with minimal manual intervention. This platform features (a) a 90 Pogo pin interface for digital microfluidic control, (b) an integrated (and motorized) photomultiplier tube for chemiluminescent detection, and (c) a magnetic lens assembly which focuses magnetic fields into a narrow region on the surface of the DMF device, facilitating up to eight simultaneous digital microfluidic magnetic separations. The new platform was used to implement a three-level full factorial design of experiments (DOE) optimization for thyroid-stimulating hormone immunoassays, varying (1) the analyte concentration, (2) the sample incubation time, and (3) the sample volume, resulting in an optimized protocol that reduced the detection limit and sample incubation time by up to 5-fold and 2-fold, respectively, relative to those from previous work. To our knowledge, this is the first report of a DOE optimization for immunoassays in a microfluidic system of any format. We propose that this new platform paves the way for a benchtop tool that is useful for implementing immunoassays in near-patient settings, including community hospitals, physicians' offices, and small clinical laboratories.

A Microfluidic DNA Library Preparation Platform for Next-Generation Sequencing

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.