Microfluidics-based EGFR mutation detection and its implication in the resource-limited clinical setting.

Management of lung cancer today obligates a mutational analysis of the epidermal growth factor receptor (EGFR) gene particularly when Tyrosine Kinase Inhibitor (TKI) therapy is being considered as part of prognostic stratification. This study evaluates the performance of automated microfluidics-based EGFR mutation detection and its significance in clinical diagnostic settings. Formalin-fixed, paraffin-embedded (FFPE) samples from NSCLC patients (n = 174) were included in a two-phase study. Phase I: Validation of the platform by comparing the results with conventional real-time PCR and next-generation sequencing (NGS) platform. Phase II: EGFR mutation detection on microfluidics-based platform as part of routine diagnostics workup. The microfluidics-based platform demonstrates 96.5% and 89.2% concordance with conventional real-time PCR and NGS, respectively. The system efficiently detects mutations across the EGFR gene with 88.23% sensitivity and 100% specificity. Out of 144 samples analysed in phase II, the platform generated valid results in 94% with mutation detected in 41% of samples. This microfluidics-based platform can detect as low as 5% mutant allele fractions from the FFPE samples. Therefore the microfluidics-based platform is a rapid, complete walkaway, with minimum tissue requirement (two sections of 5 µ thickness) and technical skill requirement. The method can detect clinically actionable EGFR mutations efficiently and can be considered a reliable diagnostic platform in resource-limited settings. From receiving samples to reporting the results this platform provides accurate data without much manual intervention. The study helped to devise an algorithm that emphasizes effective screening of the NSCLC cases for EGFR mutations with varying tumour content. Thus it helps in triaging the cases judiciously before proceeding with multigene testing.

Comparative Assessment of DNA Extraction Techniques From Formalin-Fixed, Paraffin-Embedded Tumor Specimens and Their Impact on Downstream Analysis.

OBJECTIVES: Good-quality nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) specimens remains a challenge in molecular-oncopathology practice. This study evaluates the efficacy of an in-house developed FFPE extraction buffer compared with other commercially available kits. METHODS: Eighty FFPE specimens processed in different surgical pathology laboratories formed the study sample. DNA extraction was performed using three commercial kits and the in-house developed FFPE extraction buffer. DNA yield was quantified by a NanoDrop spectrophotometer and Qubit Fluorometer, and its purity was measured by the 260/280-nm ratio. A fragment analyzer system was used for accurate sizing of DNA fragments of FFPE DNA. The downstream effects of all extraction methods were evaluated by polymerase chain reaction (PCR) and Sanger sequencing. RESULTS: In comparison with the commercial kits, the in-house buffer yielded higher DNA quantity and quality number (P < .0001). In addition, DNA integrity and fragment size were preserved in a significantly greater number of samples isolated with the in-house buffer (P < .05). The target PCR amplification rate with the in-house buffer extracted samples was also significantly higher, with 98% of the samples showing interpretable sequencing results. CONCLUSIONS: The in-house developed FFPE extraction buffer performed superior to other methods in terms of suitability for downstream applications, time, cost-efficiency, and ease of performance.

Impact of COVID-19 on quality checks of solid tumor molecular diagnostic testing-A surveillance by EQAS provider in India.

BACKGROUND: Molecular tests in solid tumours for targeted therapies call for the need to ensure precision testing. To accomplish this participation in the External Quality Assessment Program (EQAS) is required. This evaluates the consistency of diagnostic testing procedures and offers guidance for improving quality. Outbreak of COVID-19 pandemic led to worldwide lockdown and disruption of healthcare services including participation in EQAS.The present study describes the extended scope of EQAS offered byMPQAP (Molecular Pathology Quality Assurance Program), the first proficiency test provider for solid tumor diagnostics in India. The study surveys the preparedness of molecular testing laboratories in routine diagnostics and participation for quality assessment scheme. METHODS: A documented guideline for measures and precautions to be carried by testing laboratories in performing routine diagnostic tests during the lockdown period were charted and distributed to all MPQAP participant centres. A survey was conducted for MPQAP participants to check whether laboratories were involved in COVID-19 testing and to evaluate the impact of lockdown on the operations of diagnostics procedures. From the acquired response of the survey, 2 cycles out of initially proposed 11 cycles were executed with transformed approach using digital tools and image interpretation modules. FINDINGS: Out of 25 solid tumour testing laboratories registered as participants, 15 consented to participate in survey. The summary of survey conveyed the impact of COVID-19onroutine operations of diagnostics tests such as shortcomings in inventory and human resource management. Thirteen participants showed active willingness and consented to participate in EQAS test scheme. INTERPRETATIONS: The survey findings and assessment of EQAS cycles endorsed the quality testing procedures carried by participating laboratories throughout the lockdown. It highlighted the utility of EQAS participation during pandemic along with emphasis on safety measures for continual improvement in quality of diagnostic services.

Liquid Drug Dosage Measurement Errors with Different Dosing Devices.

An observational study was carried out to determine the magnitude of dosing errors made by parents, the most-preferred drug delivery device and the association of age, gender, education of the caregiver and number of children with the proportion of accurate doses. After enrolment, parents of children aged 6-60 mo were instructed to measure 5 ml of syrup paracetamol using any of the devices (stainless steel spoon, disposable plastic syringe, dosing cup with etched markings) displayed. The quantum of measured dose was confirmed using a calibrated glass cylinder. Error was defined as over 10% variation around the prescribed dose. Of 386 participants, 72 (18.65%) committed error, with 58 (15.02%) and 14 (3.62%) committing mild and moderate errors, respectively. Measuring cup (270, 69.95%) was the commonest device chosen. Use of syringe was associated with greater accurate measurements (P < 0.05) with only 3 (3.57%) committing error compared to 18 (56.25%) and 51 (18.88%) committing error with spoon and cup, respectively. On multivariate analysis, device was the only factor significantly associated with accuracy in measurements.

Air-lift reactor system for the treatment of waste-gas-containing monochlorobenzene.

An air-lift bioreactor (ALR) system, applied for the treatment of waste-gas-containing monochlorobenzene (MCB) was seeded with pure culture of Acinetobacter calcoaceticus, isolated from soil as a starter seed. It was found that MCB was biologically converted to chloride as chloride was mineralized in the ALR. After the built up of the biomass in the ALR, the reactor parameters which have major influence on the removal efficiency and elimination capacity were studied using response surface methodology. The data generated by running the reactor for 150 days at varying conditions were fed to the model with a target to obtain the removal efficiency above 95% and the elimination capacity greater than 60%. The data analysis indicated that inlet loading was the major parameter affecting the elimination capacity and removal efficiency of >95%. The reactor when operated at optimized conditions resulted in enhanced performance of the reactor.