1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays

Abstract Background Success of real-time TaqMan PCR (qPCR) in detecting pathogen targets and quantifying pathogen load is dependent upon frequent assay monitoring. This is due to i) the high degree of complementarity needed between primers / probes and genomic targets for assay accuracy and ii) natural pathogen variation and evolution. Failure to monitor and refine may result in false negativity or under quantification. Here we present a bioinformatics tool to identify potential problems resulting from newly discovered genomic mutations in primer/probe regions. Methods The tool performs an unbiased and automated search of the NCBI database, collects relevant genomic sequences based on user-defined Taxon-ID and executes a Python program to discard synthetic sequences. A profile of primer-probe sequence complementarity to targets is then generated. While the tool can be used for any microbe, here we present results for our laboratory’s cytomegalovirus (CMV) qPCR primer-probe analysis. In addition, our laboratory’s traditional approach utilizing alignment software was performed (download of all CMV sequences (~10,000) followed by iterative alignment building of these against our primers and probes). The amount of time to perform the automated and manual methods was recorded. Results The tool retrieved 8,732 sequences from NCBI and compared these to the CMV qPCR primers and probes. The tool found 2,501 alignments between the primers / probes and the downloaded genomic data (~15 minutes to finish (6 CPUs)). A total of 64% (1,624/2,501) of BLASTn alignments were exact matches between all primers / probes and viral genomic sequences. 17.5% (439/2,501) of alignments had 1 mismatch at either 5’ or 3’ terminus, and 1% (25/2,501) of alignments had two mismatches with the primers / probes. Similar results were found using a primarily manual approach (which took approx. 5 hours computing time and 20 hours of labor). Conclusion This new bioinformatics approach performed indistinguishably vs. a manual approach and did so in minutes rather than days. Both methods led to the conclusion that, by virtue of our design involving overlapping primers and probes, none of the identified mismatches are predicted to lead to false negativity or under quantification in our current CMV qPCR assay. Disclosures All authors: No reported disclosures.

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Multimodal analgesia in bariatric surgery

Regional Anesthesia and Acute Pain Management ◽

10.18821/1993-6508-2016-10-4-254-261 ◽

2016 ◽

Vol 10 (4) ◽

pp. 254-261

Author(s):

M. I Neimark ◽

Roman V. Kiselev

Keyword(s):

Bariatric Surgery ◽

Regional Anesthesia ◽

Traditional Approach ◽

Multimodal Analgesia ◽

Pharmacological Agents ◽

Perioperative Analgesia ◽

High Degree ◽

Mandatory Use ◽

Different Levels

This review is devoted to the treatment of postoperative pain in bariatric surgery. At present, the prevalence of patients with a high degree of obesity is an epidemic that leads steadily growing number of bariatric operations. Showing the risk factors in the traditional approach to the use of opioids in these patients, as well as the consequences of inadequate analgesia in these patients. Details are presented modern pharmacological agents acting on different levels nociceptive system. Substantiates the role of a multi-modal approach to perioperative analgesia, mandatory use of regional anesthesia. The attention to the visualization neuroaxial structures using ultrasound in the context of the implementation of the efficacy and safety of regional anesthesia in patients with morbid obesity. Possible prospects for pain control in bariatric surgery.

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Efficiency of the spectral element method with very high polynomial degree to solve the elastic wave equation

Geophysics ◽

10.1190/geo2019-0087.1 ◽

2020 ◽

Vol 85 (1) ◽

pp. T33-T43

Author(s):

Chao Lyu ◽

Yann Capdeville ◽

Liang Zhao

Keyword(s):

Wave Equation ◽

Spectral Element Method ◽

Computing Time ◽

Spectral Element ◽

Two Dimensions ◽

Elastic Model ◽

Time Step ◽

High Degree ◽

Very High ◽

Element Method

The spectral element method (SEM) has gained tremendous popularity within the seismological community to solve the wave equation at all scales. Classic SEM applications mostly rely on degrees 4–8 elements in each tensorial direction. Higher degrees are usually not considered due to two main reasons. First, high degrees imply large elements, which make the meshing of mechanical discontinuities difficult. Second, the SEM’s collocation points cluster toward the edge of the elements with the degree, degrading the time-marching stability criteria and imposing a small time step and a high numerical cost. Recently, the homogenization method has been introduced in seismology. This method can be seen as a preprocessing step before solving the wave equation that smooths out the internal mechanical discontinuities of the elastic model. It releases the meshing constraint and makes use of very high degree elements more attractive. Thus, we address the question of memory and computing time efficiency of very high degree elements in SEM, up to degree 40. Numerical analyses reveal that, for a fixed accuracy, very high degree elements require less computer memory than low-degree elements. With minimum sampling points per minimum wavelength of 2.5, the memory needed for a degree 20 is about a quarter that of the one necessary for a degree 4 in two dimensions and about one-eighth in three dimensions. Moreover, for the SEM codes tested in this work, the computation time with degrees 12–24 can be up to twice faster than the classic degree 4. This makes SEM with very high degrees attractive and competitive for solving the wave equation in many situations.

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The results of external quality assessment programme on urine leukocyte and erythrocyte counting in Poland

Biochemia Medica ◽

10.11613/bm.2020.020707 ◽

2020 ◽

Vol 30 (2) ◽

pp. 278-286

Author(s):

Agnieszka Ćwiklińska ◽

Barbara Kortas-Stempak ◽

Maciej Jankowski ◽

Gabriela Bednarczuk ◽

Aleksandra Fijałkowska ◽

...

Keyword(s):

Quality Assessment ◽

External Quality Assessment ◽

Particle Analysis ◽

External Quality ◽

Automated Method ◽

Percentage Difference ◽

Target Values ◽

Important Diagnostic Tool ◽

Manual Methods

Introduction: Urine particle analysis is an important diagnostic tool. The aim of this study was to evaluate the quality of urine leukocyte (WBC) and erythrocyte (RBC) counting results obtained with manual and automated methods in Polish laboratories participating in the international external quality assessment (EQA) programme. Materials and methods: 1400 WBC and RBC counting results were obtained from 183 laboratories in EQA surveys organised by Labquality (Helsinki, Finland) from 2017 to 2019. The between-laboratory coefficient of variation (CV), the percentage difference between the laboratories' results and target values (Q-score (%)), as well as modified Youden plots were analysed. Results: For automated method groups, the medians of inter-laboratory CVs varied from 14% to 33% for WBC counting and from 10% to 39% for RBC counting. For manual method groups, the medians of CV varied from 53% to 71% (WBC) and from 55% to 70% (RBC), and they were significantly higher, in comparison to CVs for most automated method groups (P < 0.001). The highest percentage of results outside the target limits (36%) and the highest range of Q-score (%) (from - 93% to 706%) were observed for laboratories which participated in the surveys for the first or second time. The percentage of deviating results and the ranges of Q-score decreased with an increased frequency of laboratories’ participation in the surveys. Conclusions: The quality of manual methods of urine WBC and RBC counting is unsatisfactory. There is an urgent need to take actions to improve laboratories’ performance and to increase harmonisation of the results.

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Development of a semi‐automated method for tumour budding assessment in colorectal cancer and comparison with manual methods

Histopathology ◽

10.1111/his.14574 ◽

2021 ◽

Author(s):

Natalie C Fisher ◽

Maurice B Loughrey ◽

Helen G Coleman ◽

Melvin D Gelbard ◽

Peter Bankhead ◽

...

Keyword(s):

Colorectal Cancer ◽

Automated Method ◽

Manual Methods ◽

Tumour Budding

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DISCURSIVE MATRIX OF THE LANGUAGES WITH DIFFERENT SYSTEMS IN TYPOLOGICAL ASPECT

Известия СОИГСИ ◽

10.46698/w5492-6711-5209-k ◽

2020 ◽

Author(s):

Л.Т. КАЛАБЕКОВА ◽

Т.А. ТАКОЕВА

Keyword(s):

Speech Acts ◽

Traditional Approach ◽

Significant Group ◽

Surrounding World ◽

European Area ◽

Language Studies ◽

Structural Identity ◽

The Status ◽

Internal Properties ◽

High Degree

На сегодняшнем этапе развития науки контрастивная лингвистика, являясь одним из наиболее перспективных направлений в современном языкознании, все больше обращается к тексту, к речевым актам, к конкретному воплощению языковых средств в дискурсе, руководствуясь наиболее приоритетной тематикой и продуктивными методами современного лингвистики. Так, в частности, любой аспектологический анализ языкового материала представляется неполным, если не упомянуть о дискурсной теории вида, которая имеет как своих приверженцев, так и ярых оппонентов. Между тем, сторонники дискурсного анализа не противопоставляют свою концепцию традиционному подходу, а толкуют ее как расширение и дополнение последнего. Дискурсная информация близка синтаксической. Не являясь непосредственным отражением свойств окружающего мира, она лишь подтверждает статус языковой единицы в тексте. Упомянутая теория предполагает связывание значений аспектуальных граммем не с внутренними свойствами самих ситуаций, описываемых глагольной лексикой, а с их функциональной значимостью в пределах конкретного текста. Появление у видовой граммемы дискурсных употреблений рассматривается как признак ее языковой «зрелости» и высокой степени грамматикализованности. В языках с разными структурными и внутриязыковыми установками (каковыми являются осетинский, русский и английский) средства репрезентации характера протекания действия принципиально различны. При этом немаловажным в осмыслении слагающихся межъязыковых коллизий становится этнический компонент: существенно неодинаковое ментальное восприятие разными этносами окружающего мира, их способность по-разному отображать одни и те же явления действительности. Вместе с тем сегодня постулируется идея существования общеевропейского языкового стандарта, согласно которой в пределах европейского ареала можно обозначить значительную группу языков, объединенных общностью их структурной идентичности, когда ареальная близость языковых культур оказывает влияние не только на инвентарь грамматических значений, но и на принципы устройства грамматических систем. At the current stage of science development, contrastive linguistics, as one of the most promising fields in modern linguistics, increasingly turns to the text, to speech acts, to the realization of linguistic means in discourse, using the most productive methods and the topic which is given the highest priority of contemporary language studies. Thus, in particular, any aspectological analysis of language material seems incomplete without mentioning the discourse theory of aspect, which has both its adherents and ardent opponents. Meanwhile, the adherents of discourse analysis do not oppose their concept to the traditional approach, but interpret it as its extension and complement. Discourse information is close to the syntactic one. It only confirms the status of the language unit in the text, but does not reflect the properties of the surrounding world directly. The mentioned theory assumes linking the values of aspectual grammars not with the internal properties of the situations described by the verbal vocabulary, but with their functional significance within a specific text. The appearance of discursive uses in a specific grammeme is considered as a sign of its linguistic "maturity" and a high degree of grammaticalization. In languages with different structural and intra-linguistic settings (such as Ossetian, Russian, and English), the means of representing the nature of the action flow are fundamentally different. At the same time, the ethnic component becomes important in understanding the resulting interlanguage conflicts: significantly different mental perception of the surrounding world by different ethnic groups, their ability to display the same phenomena of reality in different ways. At the same time, the idea of the existence of a pan-European language standard is postulated today. According to this idea a significant group of languages united by the commonality of their structural identity can be designated within the European area. In this language group, the areal proximity of linguistic cultures affects not only the inventory of grammatical meanings, but also the principles of the grammatical systems structure.

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Development of a semi-automated method for tumor budding assessment in colorectal cancer and comparison with manual methods

10.1101/2021.06.17.448482 ◽

2021 ◽

Author(s):

Natalie C Fisher ◽

Maurice B Loughrey ◽

Helen G Coleman ◽

Melvin D Gelbard ◽

Peter Bankhead ◽

...

Keyword(s):

Clinical Pathology ◽

Tumor Budding ◽

Strongly Correlated ◽

Colon Cancers ◽

Automated Method ◽

Discrete Area ◽

Prognostic Feature ◽

Manual Methods ◽

Automate Assessment ◽

Tumor Edge

Tumor budding is an established prognostic feature in multiple cancers but routine assessment has not yet been incorporated into clinical pathology practice. Recent efforts to standardize and automate assessment have shifted away from haematoxylin and eosin (H&E)-stained images towards cytokeratin (CK) immunohistochemistry. In this study, we compare established manual H&E and cytokeratin budding assessment methods with a new, semi-automated approach built within the QuPath open-source software. We applied our method to tissue cores from the advancing tumor edge in a cohort of stage II/III colon cancers (n=186). The total number of buds detected by each method, over the 186 TMA cores, were as follows; manual H&E (n=503), manual CK (n=2290) and semi-automated (n=5138). More than four times the number of buds were detected using CK compared to H&E. A total of 1734 individual buds were identified both using manual assessment and semi-automated detection on CK images, representing 75.7% of the total buds identified manually (n=2290) and 33.7% of the total buds detected using our proposed semi-automated method (n=5138). Higher bud scores by the semi-automated method were due to any discrete area of CK immunopositivity within an accepted area range being identified as a bud, regardless of shape or crispness of definition, and to inclusion of tumor cell clusters within glandular lumina ('luminal pseudobuds'). Although absolute numbers differed, semi-automated and manual bud counts were strongly correlated across cores (ρ=0.81, p<0.0001). Despite the random, rather than 'hotspot', nature of tumor core sampling, all methods of budding assessment demonstrated poorer survival associated with higher budding scores. In conclusion, we present a new QuPath-based approach to tumor budding assessment, which compares favorably to current established methods and offers a freely-available, rapid and transparent tool that is also applicable to whole slide images.

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Total and direct-reacting bilirubin values by automated methods compared with liquid chromatography and with manual methods for determining delta bilirubin

Clinical Chemistry ◽

10.1093/clinchem/36.5.788 ◽

1990 ◽

Vol 36 (5) ◽

pp. 788-791 ◽

Cited By ~ 2

Author(s):

P Rosenthal ◽

M T Keefe ◽

D Henton ◽

M Cheng ◽

C R Lee ◽

...

Keyword(s):

Liquid Chromatography ◽

Bile Acids ◽

Total Bilirubin ◽

Clinical Use ◽

Serum Samples ◽

Manual Method ◽

Automated Method ◽

Manual Methods ◽

Automated Methods ◽

Conjugated Bilirubin

Abstract This study compares total and direct-reacting bilirubin values in 40 serum samples from patients with various diagnoses, as measured by automated methods (Beckman Synchron CX-5, Beckman Astra 8, Kodak Ektachem 700) and HPLC and by a manual method for delta bilirubin. For total bilirubin, within-run CVs were less than 6%. The Ektachem 700 method underestimated bilirubin with serum samples from patients with Crigler-Najjar syndrome and from newborns in whom unconjugated bilirubin concentrations were increased but conjugated bilirubins were not present or were present only in small amounts. The Astra 8 and Synchron CX-5 methods were inaccurate with cholestatic serum samples, in which conjugated bilirubin concentrations were increased and other compounds such as bile acids could be expected to interfere. We conclude that each automated method examined provides reasonable estimates for total and direct-reacting bilirubin values for routine clinical use. The need for each laboratory to select the appropriate bilirubin method for its particular situation is obvious.

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Comparative Analysis of Erythrocyte Sedimentation Rate Measured by Automated and Manual Methods in Anaemic Patients

Journal of Laboratory Physicians ◽

10.1055/s-0040-1721155 ◽

2020 ◽

Vol 12 (04) ◽

pp. 239-243

Author(s):

Vikram Narang ◽

Sumit Grover ◽

Amandeep Kaur Kang ◽

Avantika Garg ◽

Neena Sood

Keyword(s):

Erythrocyte Sedimentation Rate ◽

Sedimentation Rate ◽

Manual Measurement ◽

Automated Method ◽

Erythrocyte Sedimentation ◽

Manual Methods ◽

Automated Instrument ◽

Very High ◽

Edta Blood ◽

High Range

Abstract Purpose Erythrocyte sedimentation rate (ESR) is a widely used indicator of inflammation and a routinely done hematology investigation to monitor patients of autoimmune and infectious diseases. We aimed to compare the ESR results obtained by Roller 20LC automated instrument and standard reference Westergren method and analyzed the effect of anemia (hematocrit) on ESR measurements through the automated method. Methods We analyzed 1377 random anemic OPD patients (hematocrit [HCT] < 35%) for ESR levels measured by Roller 20LC using EDTA blood and Westergren method using citrated blood for a one and half year period from January 1, 2018 to June 30, 2019. Fabry’s formula was used to correct the Westergren ESR. Results The total number of samples after evaluation were divided into low (n = 232), intermediate (n = 417), high (n = 406), and very high range of ESR (≥100 mm/hr; n = 422). Mean difference between values of corrected and automated ESR for the low, intermediate, high and very high ESR range was 2.33 ± 5.03, 10.95 ± 8.04, 28.22 ± 19.11 and 43.3 ± 19.22 mm/hr, respectively. The 95% limit of agreement calculated by the Bland–Altmann analysis between the two methods for low-ESR range was −7.53 to 12.2 (highest correlation coefficient –0.65), while for very high ESR, range was −5.1 to 81.5 (least coefficient of 0.18) (p < 0.001). Conclusion In laboratories with high-sample load and where manual measurement may be tedious, the automated method of ESR measurement can safely replace the Westergren method for low-ESR values in patients with low hematocrit. While for high-ESR values, validation by the standard Westergren method may be needed.

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"True" Creatinine Chromogen Determination in Serum and Urine by Semi-automated Analysis

Clinical Chemistry ◽

10.1093/clinchem/11.8.763 ◽

1965 ◽

Vol 11 (8) ◽

pp. 763-770 ◽

Cited By ~ 14

Author(s):

E Polar ◽

J Metcoff

Keyword(s):

Cation Exchange ◽

Phosphate Buffer ◽

Exchange Resin ◽

Cation Exchange Resin ◽

Automated Analysis ◽

Positive Error ◽

Strong Cation Exchange ◽

Automated Method ◽

Manual Methods ◽

Serum And Urine

Abstract The Jaffè reaction, used to determine creatinine in urine and plasma or serum, gives a positive error in the latter because of the interference of noncreatinine. In man (particularly children) endogenous creatinine normally occurs at a relatively low concentration in serum (0.3-1.0 mg./100 ml.). The analytic error contributed by noncreatinine chromogens, therefore, may be considerable. Of various manual methods to eliminate this none has been used with automated creatinine analyses, which generally measure total creatinine-like chromogen. In the present investigation a strong cation-exchange resin is used manually for rapidly adsorbing and desorbing "true" creatinine from plasma which is then analyzed by an automated method. A phosphate buffer (pH 12.4) is substituted for 5% (w/v) NaOH, dialysis eliminated, and the instrument manifold design is modified. Concentrations of "true" creatinine from 0.2-2.0 mg./100 ml. can be determined at a rate of 20 samples per hr. in serum or 40/hr. in urine, with a recovery of 98-101%.

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AFLP analysis of genetic polymorphism and evolutionary relationships among cultivated and wild Nicotiana species

Genome ◽

10.1139/g01-060 ◽

2001 ◽

Vol 44 (4) ◽

pp. 559-571 ◽

Cited By ~ 35

Author(s):

Nan Ren ◽

Michael P Timko

Keyword(s):

Genetic Polymorphism ◽

Geographic Origin ◽

Oligonucleotide Primer ◽

Aflp Analysis ◽

Nicotiana Species ◽

Nicotiana Tabacum L ◽

Polymorphic Bands ◽

Additional Support ◽

High Degree ◽

Degree Of Similarity

Amplified fragment length polymorphism (AFLP) analysis was used to determine the degree of intra- and inter-specific genetic variation in the genus Nicotiana. Forty-six lines of cultivated tobacco (Nicotiana tabacum L.) and seven wild Nicotiana species, including N. sylvestris, N. tomentosiformis, N. otophora, N. glutinosa, N. suaveolens, N. rustica, and N. longiflora, were analyzed, using at least eight different oligonucleotide primer combinations capable of detecting a minimum of 50 polymorphic bands per primer pair. The amount of genetic polymorphism present among cultivated tobacco lines (N. tabacum) was limited, as evidenced by the high degree of similarity in the AFLP profiles of cultivars collected worldwide. Six major clusters were found within cultivated tobacco that were primarily based upon geographic origin and manufacturing quality traits. A greater amount of genetic polymorphism exists among wild species of Nicotiana than among cultivated forms. Pairwise comparisons of the AFLP profiles of wild and cultivated Nicotiana species show that polymorphic bands present in N. tabacum can be found in at least one of three proposed wild progenitor species (i.e., N. sylvestris, N. tomentosiformis, and N. otophora). This observation provides additional support for these species contributing to the origin of N. tabacum.Key words: AFLP, evolution, genetic diversity, Nicotiana, tobacco, wild relatives of tobacco.

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