Prep Method Research Articles

Appraising the HIV Prevention Cascade methodology to improve HIV prevention targets: Lessons learned from a general population pilot study in east Zimbabwe.

Multiple HIV Prevention Cascades (HPC) formulations have been proposed to assist advocacy, monitoring of progress of HIV prevention implementation and research to identify ways to increase use of HIV prevention methods. Schaefer and colleagues proposed a unifying formulation suitable for widespread use across different populations which could be used for routine monitoring or advocacy. Robust methods for defining and interpreting this HPC formulation using real world data is required. Data collected as part of the Manicaland Pilot HIV Prevention Cascades Study, east Zimbabwe, in 2018-19, was used to validate the HPC framework for PrEP, VMMC, male condom and combination prevention method use. Validation measures included feasibility of populating the HPC, contrasting simple vs complex measures of the HPC (using 2-sample proportion test), and testing ability of main bars to predict prevention use and testing whether sub-bars explained why people were lost from the HPC using logistic regression. It was possible to populate the HPC for both individual and combined prevention methods using pilot survey data. Most steps were associated with prevention method usage outcomes, except for VMMC. There were significant overlaps between individuals reporting positive responses for the main bar and those citing barriers to motivation. To refine the HPC's access bar definition, it is suggested to also consider individuals who report access barriers. While the HPC framework identifies barriers to individual prevention methods, challenges arise in identifying those for combined prevention. Our study successfully utilised questionnaires from the Manicaland HPC pilot survey to measure the HPC for individual and combined prevention methods. This demonstrates the feasibility of populating this framework using general population survey data and designated questionnaire modules. We propose a final formulation of the HPC, questionnaire modules and methods to create it. With proper evaluation and promotion, the HPC can enhance prevention services, aiding in the crucial reduction of HIV incidence.

Urinalysis and culture results of free-catch urine samples in dogs: a randomised controlled trial.

To evaluate the prevalence of bacterial presence in free-catch urine samples preceded by either a standardised prepped ("clean-catch") protocol versus unprepped (non-cleaned) voiding. The study was a single-centre prospective single-blinded randomised controlled trial. Urine samples were obtained from 100 client-owned dogs presenting for routine evaluation. Dogs were randomly assigned to either the prepped group (preputial or peri-vulvar area cleaned with sterile saline before collection) or the unprepped group (no preliminary cleansing) stratified by sex. Urinalysis and urine culture (blood and MacConkey agar) were performed on all samples. Significant bacterial presence on urine culture was defined as >104 colony forming units (CFU)/mL. There were no statistically significant associations between prepped versus unprepped collection method or sex with a urinalysis positive for bacteriuria. However, on culture, significant bacterial growth was almost five times more likely to be associated with males relative to females (odds ratio 4.59, 95% confidence interval 1.61 to 13.10). The probability of finding a positive culture was not statistically associated with prep method (odds ratio 1.43, 95% confidence interval 0.50 to 4.08). For the majority of dogs without clinical signs of urinary tract infection, free-catch urine collection does not result in significant bacteriuria found on analysis or culture. The presence of bacteria found in free-catch samples may be secondary to sample contamination or subclinical bacteriuria. Sample contamination or subclinical bacteriuria may be more prevalent in male dogs.

Abstract 326: Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method

Abstract In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient surgical samples, where in most cases high quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and highly variable damage from fixation, storage, and extraction methods. It is difficult to obtain libraries with sufficient coverage and the sequencing artifacts arising from damaged DNA bases confound somatic variant detection. Additionally, many laboratories process FFPE tumor samples alongside matched, high quality, normal DNA and many library prep workflows are not readily compatible with both sample types. We developed a novel NGS library prep method compatible with both high quality and very low quality FFPE DNA samples, employing a novel enzymatic DNA repair mix, enzymatic fragmentation mix, and PCR master mix. To validate this workflow on real patient sample sets, we obtained DNA extracted from matched tumor and normal tissue of various tissue types preserved by both fresh frozen and FFPE methods. The FFPE DNA samples ranged in quality from DNA integrity number (DIN) 1.5 to 6.8, including samples stored for nearly 3 decades. We prepared libraries using this method compared against other library prep workflows and sequenced them by WGS and target capture. We assessed the performance of these workflows by library yield, library quality metrics, depth and evenness of coverage, and somatic variant calling with fresh frozen-extracted DNA providing the gold standard for library quality and mutation content. This new enzymatic fragmentation-based library prep workflow not only reduced the false positive rate in somatic variant detection by repairing damage-derived mutations in FFPE DNA samples but also improved the library yield, library quality metrics (including mapping, chimeras, and properly-paired reads), library complexity, coverage depth and uniformity, and hybrid capture library quality metrics. Comparing the variant calls from matched FFPE and frozen tissues revealed an improved sensitivity and accuracy of variant calling using this library prep method compared to mechanical shearing and other enzymatic fragmentation library prep approaches. This new suite of enzyme mixes allows even highly damaged FFPE samples to achieve high quality libraries with a greater sensitivity for somatic variant identification. The workflow is robust and flexible, compatible with both FFPE DNA and matched high quality DNA samples as well as automation-friendly for convenience in sample processing. Citation Format: Margaret R. Heider, Jian Sun, Brittany Sexton, Bradley W. Langhorst, Laura Blum, Pingfang Liu. Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 326.

B-232 Versatile, Extraction-Free Nucleic Acid Sample Preparation in 15 Seconds Via Hyperbaric Heating

Abstract Nucleic acid extraction-based PCR sample prep is currently limited by long processing times, liquid transfers, kit cost, and lab storage requirements. Extraction-free approaches enable streamlined sample prep, but lack sensitivity compared to extraction-based methods. We studied several sample types to demonstrate the effectiveness of a novel 15-second Hyperbaric Heating (HBH) process to prepare nucleic acids for PCR. HBH exposes the sample to a short duration, high temperature environment to effectively lyse organisms and denature PCR inhibitors such as nucleases in seconds. Samples are introduced and sealed in a pressure-tight metallic vessel, which is then heated in seconds using magnetic induction. Within the sealed metallic vessel, the vaporization of the aqueous sample raises the internal pressure, enabling an internal temperature higher than 100°C to be achieved. Chelating agents and other proteins in HBH also protect the target nucleic acids from fragmentation. Post HBH, the sample is ready for direct PCR amplification without further dilution or extraction. Efficacy of HBH for efficient PCR amplification was tested against commercial kits. HBH was applied to viral, bacterial, eukaryotic, and fungal organisms. For SARS-CoV-2, HBH detected down to 250 viral copies/mL. Additionally, HBH outperformed commercial kits for PCR sample prep of Candida albicans and Bacillus subtilis. HBH is a generalizable sample prep technology with sensitivity comparable to that of current extraction kits. We have applied HBH to a multitude of organisms and have demonstrated that HBH can be employed as a rapid and comprehensive nucleic acid prep approach for applications that involve PCR amplification post sample prep. Future integration of HBH in devices suitable for point of care molecular testing are underway. We also envision HBH to be a useful, generalizable sample prep method for the molecular lab in applications where cost, simplicity, and speed are essential for preparing nucleic acids for amplification.

A-376 Development of a 10-Minute Sample to Answer RT-PCR Respiratory Panel Performed on Point of Care (POC)

Abstract Background The COVID-19 pandemic has shown the importance of fast and sensitive PCR testing to identify SARS-CoV-2 (SC2) infected patients. Identifying infected individuals with a fast sample to answer test provides the opportunity to limit the spread of the virus and prescribe antimicrobial treatment in the same visit. Additionally, the post pandemic world has seen simultaneous outbreaks of RSV, influenza, and SC2, the so-called tridemic. These higher risk respiratory viruses can appear against a background of lower risk viruses such as rhinovirus, adenovirus, enterovirus, other corona viruses, and bacterial infections that present with similar symptoms and make triage challenging. Fast turnaround molecular testing along with multiplex detection of all key pathogens associated with flu-like symptoms is important for timely selection of treatment, counseling to contain the spread of disease, and provide public health officials information on prevalence of outbreaks. AMDI is developing a cloud-based, ultrafast, fully automated POC molecular technology that enables detection of up to 32 microbial targets in less than 10 min from sample to answer. A 15-second extraction-free hyperbaric heating (HBH) step is performed on the input swab sample to release nucleic acid and neutralize PCR inhibitors in the sample in seconds. The sample is directly transferred without dilution to a chamber where it mixes with dry RT-PCR reagents and then is dispatched to eight thin (<250 µm thick) PCR reaction cuvettes where rapid heat transfer can occur. This is followed by thermocycling on an instrument designed to achieve 10 sec per cycle and detect four fluorescent signals per cuvette. This study first evaluates the HBH method against nucleic acid purification assays using SC2 positive clinical samples. Next, a preliminary LoD on the AMDI system (HBH and fast thermocycling) was performed using inactivated SC2 and RSV whole organism in a nasal swab matrix. Methods To evaluate HBH as a sample prep method, 12 known SC2 positive remnant clinical samples in VTM or saline were diluted 1:1 with AMDI collection buffer, treated by HBH, tested by RT-PCR on QuantStudioTM and compared with the Roche LIAT. A preliminary SC2 and RSV LoD on the full AMDI system was determined by spiking dilutions of inactivated viruses into pooled nasal swab matrix, followed by treatment with HBH, and testing by RT-PCR using the ultra-fast thermocycler. Results In clinical sample testing HBH shows equal or greater sensitivity than LIAT, even for high Ct samples. HBH followed by RT-PCR detected 12/12 (100% sensitivity) samples while Roche LIAT detected 11/12 (92% sensitivity). When HBH is coupled with AMDI ultra-fast PCR thermocycling technology, an SC2 preliminary LoD of 500 copies/mL and an RSV preliminary LoD of 250 copies/mL was obtained in <9 min including a 60 second RT step. Conclusion HBH is an efficient and effective sample prep method and when coupled with ultrafast thermocycling provides sensitive PCR results in 10 minutes sample to answer.

Comparison of liquid-based cytology and conventional preparations in nongynecological cytology

Abstract Introduction: The liquid-based cytology (LBC) has shown significant advantages over conventional preparation in cervical cytology. The use of LBC in aspiration and exfoliative nongynecologic cytology has been on the rise since the last few years with relatively few studies being available depicting its role in fine-needle aspiration cytology (FNAC) from other sites. The study aimed to determine the diagnostic efficacy of LBC in nongynecological exfoliative as well as FNAC samples and compare the conventional and LBC methods for cytomorphological differences. Materials and Methods: This is a cross-sectional, analytical study that included the samples collected from two tertiary care centers over 8 months (January 2022 to August 2022). A total of 168 consecutive samples of aspiration and exfoliative nongynecological cytology were included. In each case, two passes were carried out one for a conventional smear (CS) and the other for LBC by Eziprep/Thin Prep (TP) method followed by smear staining with H and E stain and Leishman’s stain. The slides were studied by two observers for cellularity, background (blood and necrotic cell debris), informative background (such as colloid, stromal fragments, etc.), presence of monolayer cells, and cell architecture with nuclear and cytoplasmic details by a semiquantitative scoring system. Statistical analysis was performed using Wilcoxon signed-rank test on the SPSS program and histopathology correlation was attempted wherever available by using OpenEpi software version 3. Results: The mean age was 40 years, with the majority being females (70%). The TP smears were qualitatively superior to CS with statistical differences observed in fluid cytology. There was no statistical difference observed at other sites; except TP being superior to CS with regard to the background in breast cytology. The arithmetic mean in TP was higher than CS in all the parameters. Conclusions: The cytology smears of TP were qualitatively superior to CS in aspiration and nongynecological cytology. Overall diagnostic accuracy was comparable in both techniques. The core competencies addressed in this article are: Medical knowledge, Procedural skills, Practice-based learning and improvement, and Professionalism.

Đánh giá một số sinh phẩm và phương pháp tách chiết ADN vi khuẩn Mycobacteria trên mẫu bệnh phẩm thu thập từ bệnh nhân nhiễm lao

Tuberculosis is still burden in developing countries, including Viet Nam. Molecular techniques such as PCR, Real-time PCR play an important role in tuberculosis diagnostics. Specimens for tuberculosis testing are complex such as sputums, gastric fluids. DNA extraction are commonly performed by commercial kits, with little study about efficacy for tuberculosis testing assays. This study describes efficacy of three kits G-Spin (Intron-Korea), RIBO- sorb (Amplisens-Russia), DNA/RNA Prep (Sacace-Italy), and Salting-out method using basic reagents. Samples were 15 sputums collected from tuberculosis infected cases. As the result, recovered ADN was highest in DNA/RNA Prep, folowed by Salting-out, G-Spin, and last by RIBO - sorb. Oder of purification value was RIBO - sorb, G-Spin, Salting-out, and DNA/RNA Prep. About PCR for 16S gene, 15/15 samples were succeed in G-Spin and DNA/RNA Prep, 14/15 from RIBO-prep, 13/15 from Salting-out. Real-time PCR Mycobacterium tuberculosis (Sacace-Italy) detected 14/15 samples from G-Spin, DNA/RNA Prep, and RIBO-sorb, but Salting-out detected 13/15 samples. Therefore, G-Spin and DNA/RNA Prep show higher efficacy in amplication of 16S gene and Real-time PCR assays for tuberculosis. RIBO- sorb has equal efficacy in Realtime PCR, but more priority in purification. Salting-out perform equally in recovery and purification with commercial kits, but limited in efficacy of Mycobacteria genome amplification.

Analysis of technological processes of production of spherical powders and granules of NiAl nickel monoaluminide for the needs of domestic engine building

The article is devoted to the study of the features of various methods for obtaining granules of nickel aluminide NiAl. The problems hindering the widespread use of nickel aluminide NiAl in modern aircraft and engine construction are analyzed. It has been revealed that the main problems hindering the widespread industrial use of nickel aluminide NiAl are practically zero plasticity of the material during pressure treatment and difficulties in machining parts made of this material. However, this problem can be solved with the use of pellet metallurgy technologies, when by sintering the granular material, an almost finished product is obtained that requires minimal amounts of subsequent machining. Within the framework of the conducted studies, the quality criteria of the obtained granules were determined, which include the sphericity of the granules, the stability of the obtained dimensions of the granular material, the absence of defects in the form of pores, the absence of satellites on the surface of the granules, the presence of a finely dispersed dendritic structure of the granule material. Several methods of obtaining granules of nickel aluminide NiAl have been investigated from the point of view of obtaining the highest quality raw materials, namely: the method of spraying the melted billet with a high-temperature inert gas flow (gas atomization method), the method of centrifugal spraying of the melted electrode (PREP method), the method of centrifugation of the melt using a perforated crucible. It is determined that the optimal way to obtain a high-quality granulate of NiAl material is the method of centrifugal spraying of the melted electrode. In the course of the conducted research, it was proved that the main parameter of the process of centrifugal spraying of the melted electrode, affecting the quality of the obtained granules, their diameter and the value of the dendritic parameter of the microstructure of the granules, is not so much the current strength I as the rotation speed of the melted electrode n. The optimal values of the electrode rotation speeds are determined, which are n ≈ 15000-16000 revolutions per minute at a current strength I ≈ 1000-1500 A. A technology for obtaining high-quality NiAl material granulate has been developed and tested, which includes operations for obtaining initial NiAl blanks by self-propagating high-temperature synthesis, subsequent remelting of semi-finished products, heat treatment, separation of granules and subsequent granulation by the PREP method.

Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4–6 bacterial isolates per sequencing run (20–26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

Sexual Behavior and HIV / AIDS Prevention in Male Sexual Men (MSM) in Denpasar City

HIV / AIDS cases among MSM (male sex men) in Indonesia from year to year have increased significantly. Meanwhile, Bali is a province with many HIV / AIDS cases, where Denpasar is the city with the highest number of MSM. There has never been a report on sexual behavior and HIV / AIDS prevention among MSM in Denpasar. Therefore, the researchers wanted to explore the problems of sexual behavior and its prevention in MSM in Denpasar. Purpose: This study aims to obtain a vision of sexual behavior and HIV / AIDS prevention among MSM groups in Denpasar City. This study used a qualitative design with a phenomenological approach to digging deeper into sexual behavior and HIV / AIDS prevention among MSM in Denpasar City. The selection of informants in this study was taken using convenience sampling technique and conducted in-depth interviews with 10 MSM who were productive and communicative in Denpasar. After collecting data in the field, the data will be analyzed thematically. In sexual intercourse behavior, most of the MSM prioritized variations and sensations during sexual intercourse to get sexual satisfaction, such as having group sex, using arousal-enhancing drugs, and violence engaging during sexual intercourse. Some of the informants only had one permanent sexual partner. However, there are still MSM who have more than one sexual partner and receive payment for sexual intercourse. Informants use condoms in sexual intercourse and with the PrEP method to avoid HIV / AIDS. It is necessary to conduct education and counseling on sexual orientation for the community, especially for adolescents looking for identity, educating MSM to reduce high-risk behavior during sexual intercourse.
 Keywords: Sexual behavior, MSM, prevention, HIV / AIDS, Denpasar

English

BACKGROUND The major cause of mortality among women is cancer of the uterine cervix. It is the second most common cancer worldwide. Pap smear is done as a method of cervical screening to detect precancerous and cancerous lesions of the cervix. Early detection of these lesions prevent the number of deaths from cervical cancer. Liquid based cervical cytology was developed to improve the diagnostic accuracy of pap smears. The main purpose of this study was to compare the profile of distribution of cervical smear cytology lesions obtained by thin prep preparation (liquid based cytology) and conventional pap method among women in the age group of 25 – 60 yrs. METHODS This is a cross sectional study done among 110 women from the outpatient department of Obstetrics and Gynaecology. Conventional and liquid based cytology smears were taken simultaneously from the same women and reported according to the 2001 Bethesda system. RESULTS Majority of the women included in this study fall in the age group of 41 - 50 years [65 (59.1 %)]. Women with a history of persistant vaginal infection were 13 (11.8 %). Out of the 110 pap smears taken by both conventional and liquid based methods, unsatisfactory smears were not detected in liquid based cytology and 6 (5.5 %) unsatisfactory smears were found in conventional pap method. In liquid based cytology, inflammatory smears were detected more (80 %), atrophic smears (2.7 %), smears with features of atypical squamous cells of undetermined significance (ASCUS) were 4.5 %, low-grade squamous intraepithelial lesion (LSIL) were 3.6 %, and smears with high-grade squamous intraepithelial lesion (HSIL) were 0.9 %. Epithelial cell abnormalities were found to be high in liquid based cytology. CONCLUSIONS Abnormal epithelial lesions were detected more in liquid based cytology (thin prep method). The number of unsatisfactory smears were found to be decreased when compared to conventional pap method. KEY WORDS Bethesda Cervical Cytology, Cervical intraepithelial lesion, Conventional Pap Smear, Liquid Based Cytology Smear

A robust broadband fat-suppressing phaser T2 -preparation module for cardiac magnetic resonance imaging at 3T.

Designing a new T2 -preparation (T2 -Prep) module to simultaneously provide robust fat suppression and efficient T2 preparation without requiring an additional fat-suppression module for T2 -weighted imaging at 3T. The tip-down radiofrequency (RF) pulse of an adiabatic T2 -Prep module was replaced by a custom-designed RF-excitation pulse that induces a phase difference between water and fat, resulting in a simultaneous T2 preparation of water signals and the suppression of fat signals at the end of the module (a phaser adiabatic T2 -Prep). Numerical simulations and in vitro and in vivo electrocardiogram (ECG)-triggered navigator-gated acquisitions of the human heart were performed. Blood, myocardium, and fat signal-to-noise ratios and right coronary artery vessel sharpness were compared against previously published adiabatic T2 -Prep approaches. Numerical simulations predicted an increased fat-suppression bandwidth and decreased sensitivity to transmit magnetic field inhomogeneities using the proposed approach while preserving the water T2 -Prep capabilities. This was confirmed by the tissue signals acquired in the phantom and the in vivo images, which show similar blood and myocardium signal-to-noise ratio, contrast-to-noise ratio, and significantly reduced fat signal-to-noise ratio compared with the other methods. As a result, the right coronary artery conspicuity was significantly increased. A novel fat-suppressing T2 -Prep method was developed and implemented that showed robust fat suppression and increased vessel sharpness compared with conventional techniques while preserving its T2 -Prep capabilities.

Validation of an Isothermal Amplification Platform for Microbial Identification and Antimicrobial Resistance Detection in Blood: A Prospective Study.

Background: Recent advances in nucleic acid amplification technique (NAAT)-based identification of pathogens in blood stream infections (BSI) have revolutionized molecular diagnostics in comparison to traditional clinical microbiology practice of blood culture. Rapid pathogen detection with point-of-care diagnostic-applicable platform is prerequisite for efficient patient management. The aim of the study is to evaluate an in-house developed, lyophilized OmiX-AMP pathogen test for the detection of top six BSI-causing bacteria along with two major antimicrobial resistance (AMR) markers of carbapenem and compare it to the traditional blood culture-based detection.Materials and methods: One hundred forty-three patients admitted to the Medical Intensive Care Unit, Narayana Hrudayalaya, Bangalore, with either suspected or proven sepsis, of either gender, of age ≥18 years were enrolled for the study. Pathogen DNA extracted from blood culture sample using OmiX pReP method was amplified at isothermal conditions and analyzed in real time using OmiX Analysis software.Results: Among the processed 143 samples, 54 were true negative, 83 were true positive, 3 were false negative, and 2 were false positive as analyzed by OmiX READ software. Gram-negative bacteria (91.3%) and gram-positive bacteria (75%) were detected with 100% specificity and 95.6% sensitivity along with the AMR marker pattern with a turnaround time of 4 hours from sample collection to results.Conclusion: OmiX-AMP pathogen test detected pathogens with 96.5% concordance in comparison to traditional blood culture. Henceforth, OmiX-AMP pathogen test could be used as a readily deployable diagnostic kit even in low-resource settings.How to cite this article: Maheshwarappa HM, Guru P, Mundre RS, Lawrence N, Majumder S, Sigamani A, et al. Validation of an Isothermal Amplification Platform for Microbial Identification and Antimicrobial Resistance Detection in Blood: A Prospective Study. Indian J Crit Care Med 2021;25(3):299–304.

Building students' Background knowledge on Reading Comprehension through PreP Method for the Tenth Grade Students at SMA Negeri 1 Tiakur

Building students' Background knowledge on Reading Comprehension through PreP Method for the Tenth Grade Students at SMA Negeri 1 Tiakur

A study of liquid-based cytology in cervical smears

Introduction: Cervical cancer is a major health problem. It ranks second in mortality affecting the world population. Cervical cancer has the advantage that it takes a long time for it to develop. During this time it can be diagnosed and treated properly. For this purpose pap smears (cervical smears) are routinely screened for pre-malignant lesions using Liquid Based Cytology. Purpose: The purpose is to study the prevalence of cervical lesions by EZI PREP method and to determine the prevalence of cervical lesions in various age groups. Material and Methods: This was a prospective study done in the Department of Pathology. All the data regarding the patient was recorded in a predefined proforma. The sample taken by using an endocervical brush was immersed in a vial and collected at a cytopathology laboratory for further processing by the EZIPREP method. Results: A total of 1500 cervical smears were taken into the study. The age group of females ranged from 20-80 years. All the satisfactory smears were evaluated. Inflammatory smears, infectious smears, and smears with epithelial cell abnormalities were identified in the present study. Bacterial vaginosis was the most common infectious condition found in the present study. The most common age group for epithelial cell abnormality in the present study was 41-50 years. The most common epithelial cell abnormality in the present study is High grade squamous intraepithelial lesion followed by Low grade squamous intraepithelial lesion. Conclusion: Out of the 1500 LBC smears studied, the majority (1134) of the smears studied were inflammatory. Epithelial cell abnormalities were found in 21 cases and the most common age group of epithelial abnormalities was the fifth decade.

Methods for reduction of oxygen content in production of powders-granules of heat-temperature nickel alloys

The sources for increase of oxygen in the mass of powders-granules produced by the PREP method are considered. Quantitative calculation of the increase in the proportion of oxygen and recommendations for reducing of the oxygen content and for further use of powders-granules are presented.

The Nature of Anomalous Particles (Granules) in Rapidly Quenched PREP Powders: I. A Multiscale Study of the Heating Zone of the Rotating Electrode Used to Obtain the Rapidly Quenched PREP Ni-Based Superalloy Powder

The uniformity of composition of rapidly quenched PREP particles of the powder of high temperature Ni-based superalloys and stainless steels is characterized by an important feature—the occurrence of anomalous particles (granules) with a significantly different content, mainly of microalloying interstitial elements, carbon and boron, as well as active carbide- and boride-forming alloying elements. A detailed multi-scale experimental study of the heating zone of the crater of the Ni-based superalloy electrode after its use to obtain rapidly quenched PREP powder was carried out in order to find the nature and mechanisms of the formation of anomalous granules. Direct nuclear physics methods of activation autoradiography on carbon, track autoradiography on boron, metallography, SEM, EDX, OIM were used. In the electrode crater, the heat-affected zone (HAZ) and the partially melted zone (PMZ) were detected. Intense migration of boron to the electrode surface due to the formation of thermal macrocracks was also revealed. The behavior of carbon is determined by the formation of a thin layer of melt on the surface of the crater. The features of the evolution of the terminal solidification region TSR and incipiently melted regions IMR, the main type of heterogeneity of the composition of the dendritic structure of Ni-based superalloys and stainless steels, are revealed. The interrelation of the evolution of these areas is established, which is a consequence of the thermodynamic principle of the reversibility of the processes of solidification and melting, respectively, in the smelting of an ingot electrode and in the process of subsequent atomization. The analysis of the influence of the behavior of boron, carbon, and the characteristics of the crater structure on the nature and mechanism of the formation of anomalous granules using the PREP method for producing rapidly quenched powder of the Ni-based superalloy is performed.

Abstract 5093: Clinical-grade NGS analytic platform with explicit negative calling to improve variant reporting across different laboratory protocols: Accuracy study

Abstract Introduction: Research-grade bioinformatic pipelines share a common workflow with a logical shortcut that infers where no variant was called, no variant exists. The complexity of NGS protocols makes this inference clinically unsound. Incomplete enrichment, or a sample with deletions in the region of interest can result in false negative errors. Clinical-grade NGS analytics should explicitly evaluate each region of clinical interest to distinguish regions with no informative variants from those with too little evidence to make a call. An ideal platform would produce reliable results from sequencers and library prep in clinical use without protocol-specific tuning. This study evaluates the performance of a novel Clinical-grade Analytic Platform designed to meet these challenges. Study Design: 189 samples from the NA12878 cell line, processed by 13 different laboratories, using 18 different protocols and 11 different instruments were sourced as raw sequencing data from public repositories. Each sample file was run through the Clinical-grade Analytic Platform to produce a cVCF result, a detailed accounting of variants present, regions with no variant present and regions with insufficient coverage across 14,626 exons in the 1043-gene PanCancer TestPanel. High Confidence TRUTH, obtained from the GIAB consortium, established that NA12878 DNA contains 598 variants and 14,230 exons with no variant across the 1043 genes. The cVCFs were compared to TRUTH to quantify performance. Results: The Clinical-Grade NGS Analytic Platform ran all 189 samples without protocol or laboratory-specific tuning, with an overall sensitivity of 95% and specificity of 99%. This performance includes samples with average read depth ranging from 18x to 10,000x, although regions with local read depths above 30x performed better. Hybridization capture-based methods generally outperformed amplicon-based methods at all read depths. Full presentation will discuss performance stratified by instrument, library prep method, local read depth, and variant type. Conclusion: The Clinical-grade Analytic Platform, Farsight Correo, showed strong performance across a broad range of read depths, instruments and library prep methods. The platform’s unique ability to distinguish between “No Variant Present” and “Insufficient Coverage” contributed significantly to this performance. Robust, cross-protocol performance is clinically valuable. It can enable clinical laboratories to expand or change NGS protocols without replacing analytics. It can enable companion diagnostic programs to include multiple sequencing protocols and partners. And finally, explicit negative variant calling can help AI and machine learning mirror the underlying biology; integrating variant presence and absence into new pathway-based models to significantly advance genomics-guided medicine. Citation Format: Edward P. Tang, Charlie C. Kim, Kristine C. Mechem, Glenda G. Anderson. Clinical-grade NGS analytic platform with explicit negative calling to improve variant reporting across different laboratory protocols: Accuracy study [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 5093.

Preparing Single-cell DNA Library Using Nextera for Detection of CNV.

Single-cell DNA sequencing is a powerful tool to evaluate the state of heterogeneity of heterogeneous tissues like cancer in a quantitative manner that bulk sequencing can never achieve. DOP-PCR (Degenerate Oligonucleotide-Primed Polymerase Chain Reaction), MDA (Multiple Displacement Amplification), MALBAC (Multiple Annealing and Looping-Based Amplification Cycles), LIANTI (Linear Amplification via Transposon Insertion) and TnBC (Transposon Barcoded) have been the primary choices to prepare single-cell libraries. TnBC library prep method is a simple and versatile methodology, to detect copy number variations or to obtain the absolute copy numbers of genes per cell.

Noninvasive paternal exclusion testing for cystic fibrosis in the first five to eight weeks of gestation

Prenatal genetic testing is not generally applicable to the very early stages of pregnancy (prior to week 8 gestation), a time period that is crucial to pregnant couples with high risk for transmission of genetic disease to their fetus. Therefore, we developed a new ultra-sensitive targeted next generation sequencing method for noninvasive haplotype-based paternal allele exclusion testing of the cystic fibrosis-associated gene, CFTR. This new method was compared to a conventional library prep and sequencing analysis method and all test results were validated by amniotic fluid testing at later stages of pregnancy. Out of 7 enrolled couples, who provided at least two blood samples (at least one week apart) for noninvasive CFTR testing, a result was obtained for 6 fetuses. Using the new hypersensitive method, all six couples (100%) received a correct diagnosis for the paternal allele as opposed to 3/6 (50%) when tested with the conventional strategy. Among 4 couples who provided just one early pregnancy blood draw for analysis, diagnosis was possible in one fetus, but only using the ultra-sensitive method. Thus, we describe a novel noninvasive CFTR screening method which demonstrates unprecedented fetal allele typing accuracy in the earliest stages of pregnancy.