Validity Of Assay Research Articles

DEVELOPMENT OF A QUANTITATIVE IMMUNOASSAY FOR SERUM HAPTOGLOBIN AS A PUTATIVE DISEASE MARKER IN THE SOUTHERN WHITE RHINOCEROS (CERATOTHERIUM SIMUM SIMUM).

Objective disease markers in the southern white rhinoceros (Ceratotherium simum simum) are in high demand. In the field, such markers are typically needed to decide whether a captured white rhinoceros is fit to cope with quarantine, transport, or both. Captive white rhinoceros have a need for unbiased biomarkers for early detection of disease. Acute phase proteins, including haptoglobin, are proteins that significantly change their plasma concentration in response to tissue perturbation or inflammation, such as that occurring during infection or neoplastic disease. Acute phase proteins are well known diagnostic tools in both human and veterinary medicine. In this study, an ELISA with commercially available anti-human haptoglobin antibodies for quantification of haptoglobin in white rhinoceros serum was developed. The validity of the haptoglobin assay and haptoglobin as a biomarker of disease was investigated with the use of serum samples from both captive and free-ranging animals with a well-described health status. The assay was precise (intra-assay and interassay reproducibility were 5.0% and 13.1%, respectively) and reliably quantified white rhinoceros haptoglobin serum concentrations consuming low volumes of sample. The assay was sensitive to the presence of free hemoglobin in the sample at levels corresponding to a visibly hemolyzed sample. Haptoglobin was readily measurable, baseline levels (in white rhinoceros with no clinical signs of disease) did not differ between genders, and a significant increase was seen in captive as well as in free-ranging white rhinoceros with inflammatory disease. Thus, haptoglobin is a positive acute phase protein in southern white rhinoceros with potential for use as an objective marker of disease.

EP375: Technical performance of a 455-gene preventative genomics assay

DNA-based screening in individuals without known risk factors potentially identifies those who may benefit from genetic counseling, early medical interventions, and/or avoidance of late or missed diagnoses. While not currently in widespread usage, technological advances in genetic analysis overcome barriers to access by enabling less labor-intensive and more cost-efficient means to discover variants of clinical importance. This study describes the technical validation of a 455-gene assay that is comprehensive both in its scope of medical conditions and variant detection capabilities. The GeneCompass test analyzes genes associated with Mendelian inheritance, susceptibilities, and pharmaceutical drug response to conditions in 12 clinical areas (eg, cardiovascular disease, oncology, psychiatry, neurology). The custom-designed next-generation sequencing (NGS) capture and bioinformatics pipelines discover multiple variant types, including single nucleotide variants, insertion/deletion (indel), larger copy number variants, and functional haplotypes (star alleles). Validation was performed against reference DNA from three sources: 1000 Genomes Project (n=3), Coriell biobank (n=15), and internal leftover blood (n=21) and saliva (n=12) specimens from a previously validated assay. Overall analytical sensitivity and specificity for single nucleotide variants were 97.57% and 99.99%, respectively, and for indels were 74.57% and 97.35%, respectively. While early data on genomic-based health screening suggests health benefits, widespread usage continues to be impeded due in part to cost, lack of payer coverage, and reluctance by medical societies or individual practitioners to adopt routine usage as a result of lack of education, comfort, and/or perceived utility. Despite barriers to access of genetic testing, when made aware of the opportunity for genomic screening, individuals express positive receptivity and understanding of genetic information. This study demonstrates validity of a NGS assay that is technically capable for screening purposes and potentially broadens access to preventative genomics usage.

Development, Validation, and Application of a High-Performance Liquid Chromatography Method for the Analysis of Dissolution Samples of Telmisartan and Rosuvastatin Calcium Double-Layer Formulation

Background: Telmisartan and rosuvastatin calcium fixed-dose combination double-layer tablet is used for hypertension and hyperlipidemia treatment. Owing to the critical problem in establishing the specificity, precision, and accuracy of the USP analysis method and simultaneous analysis method for both components, an analysis method that could be applied to researchers in the field is urgently needed. Objective: To develop and validate a reversed phase-high-performance liquid chromatography method for the quantitative analysis of dissolution samples of telmisartan and rosuvastatin calcium fixed-dose combination double-layer tablets, as a hypertension and hyperlipidemia treatment. Methods: The developed analysis method was validated according to USP Category I requirements. The validity of the quantitative assay of the dissolution test solution was determined based on the system suitability, specificity, linearity, accuracy, precision, and solution stability of the assay for the components. Results: The developed method was selective and precise. The retention times of telmisartan and rosuvastatin calcium was 8.1 min and 2.8 min, respectively, and system suitability was 0.217 and 0.17, respectively. Both components showed linearity at 10–120% concentration. The accuracy and precision were also within acceptable limits. The drugs in the dissolution samples were stable for a long time, no interaction occurred between the excipients and active pharmaceutical ingredients, and no interference was observed in the analysis. Conclusion: The quantitative analysis of the double-layered formulation of telmisartan and rosuvastatin calcium was accurate, selective, and precise. Therefore, this method can be recommended for use in the industrial quality control of telmisartan and rosuvastatin calcium formulations.

Detection and differentiation of active and inactive isoforms of coagulation factors II, VII, IX, and X in prothrombin complex concentrate by mass spectrometry

PurposeProthrombin complex concentrates (PCCs) are plasma products containing a mixture of four inactive/proactive coagulation factors. The activated forms of human coagulation factors, like Thrombin (FIIa), Convertin (FVIIa), activated Christmas factor (FIXa) and the activated Stuart-Prower factor (FXa), are impurities in PCCs. Until now no valid assay exists to differentiate the non activated proform (inactive) from active coagulation factor isoforms in PCCs in one measurement. Therefore, the aim of this study was to establish a mass spectrometry (LC-MS/MS)-based assay to address this issue in the ready to use medicinal product. MethodsBottom-up proteomics combining double digestion (Glu-C & Lys-C) and LC-MS/MS, was used to differentiate the inactive and active forms of the coagulation factors Prothrombin (FII), Proconvertin (FVII), Christmas factor (FIX) and the Stuart-Prower-factor (FX) in PCCs. Results and conclusionsA targeted pseudo-multiple reaction monitoring (pMRM-LC-MS/MS)-assay was developed for the specific detection of four different coagulation factors in PCCs. Proteotypic peptides for the inactive/active isoforms (zymogen) of the four coagulation factors were identified and validated by the investigation of six investigational and one commercially available PCCs.In conclusion, the semi-quantitative determination and the distinction between the active and the inactive isoform of the respective coagulation factors were possible in one liquid chromatography tandem mass spectrometry (LC-MS/MS) run.

The Role of Antigen Rapid Diagnostic Test in COVID-19 Diagnosis

Since the emergence of a novel infection due to the SARS-CoV-2 virus (COVID-19), the World Health Organization has urged countries to develop diagnostic tests to combat the pandemic. Molecular assays were developed following the release of the gene sequence of the virus in January 2020. Reverse transcription-quantitative PCR (RT-qPCR) is taken as the gold standard for the diagnosis of COVID-19. However, due to its limitations, highly sensitive methods for detecting antigens (antigen rapid diagnostic tests) have been developed that would help in a timely and accurate diagnosis. Antigen rapid diagnostic tests (Ag-RDTs) can help guide patient management at the point of care by random screening, re-testing, and timely decision-making in the field of public health. When the affordability and validity of the diagnostic assay are involved, no assay can show 100% correct results. Further studies need to be done to better understand the response of the Ag-RDTs in different settings. Nevertheless, Ag-RDTs can play a complementary role in the response and case management of COVID-19.

Nationwide Laboratory Network for AML Cross-Validated NGS Studies: Results from a Real-Life Cohort of the Pethema Group

Nationwide Laboratory Network for AML Cross-Validated NGS Studies: Results from a Real-Life Cohort of the Pethema Group

PSV-B-28 Late-Breaking: Development and validation of a Total Inflammation Index™ for identifying inflammation in Labrador Retrievers using a pressure walkway

Abstract The objective of this trial was to develop an index system to identify inflammation in Labrador Retrievers using a pressure walkway system. Gait analysis data can be difficult to interpret between treatment groups or for identifying low grade inflammation. To calculate the Total Inflammation Index™, the distance away from the ideal score was calculated for four parameters for each dog, including gait lameness score, total pressure index, step/stride ratio, and hind reach. These values were equally weighted and added together to produce the Total Inflammation Index™. For validation, the Total Inflammation Index™ values were compared to biomarker data for inflammation including cartilage oligomeric matrix protein, interleukin-6, creatine kinase, and c-reactive protein. Forty Labrador Retrievers (20 male/20 female) were used in this trial. All dogs were passed over the pressure walkway (Gait4Dogs; CIR Systems, Inc) to obtain gait analysis at baseline, 24h prior to the first 5km run, 24h after the first 5km run, 24h prior to the final 16km run, and 24h after the final 16km run. All biomarkers and the Total Inflammation Index™ were both significantly lower at the pre-exercise timepoints and elevated after post-exercise timepoints (P < 0.01). The Total Inflammation Index™ had significant correlation between timepoints and all biomarkers, including cartilage oligomeric matrix protein (P < 0.01), interleukin-6 (P < 0.05), creatine kinase (P < 0.01), and c-reactive protein (P < 0.05). The Total Inflammation Index™ appears to be a valid assay to evaluate generalized inflammation in Labrador Retrievers, and is in agreement with inflammatory biomarker values.

Genetic drift in the genome of SARS COV-2 and its global health concern.

The outbreak of the current coronavirus disease (COVID‐19) occurred in late 2019 and quickly spread all over the world. The severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) belongs to a genetically diverse group that mutates continuously leading to the emergence of multiple variants. Although a few antiviral agents and anti‐inflammatory medicines are available, thousands of individuals have passed away due to emergence of new viral variants. Thus, proper surveillance of the SARS‐CoV‐2 genome is needed for the rapid identification of developing mutations over time, which are of the major concern if they occur specifically in the surface spike proteins of the virus (neutralizing analyte). This article reviews the potential mutations acquired by the SARS‐CoV2 since the pandemic began and their significant impact on the neutralizing efficiency of vaccines and validity of the diagnostic assays.

MP16-15 CLINICAL VALIDATION OF URINE DNA-BASED PENK METHYLATION TEST FOR DETECTING BLADDER CANCER IN HEMATURIA PATIENTS

You have accessJournal of UrologyBladder Cancer: Non-invasive II (MP16)1 Sep 2021MP16-15 CLINICAL VALIDATION OF URINE DNA-BASED PENK METHYLATION TEST FOR DETECTING BLADDER CANCER IN HEMATURIA PATIENTS Jae Sung Lim, Bo-Ram Bang, Justin J. Lee, Ki Hak Song, TaeJeong Oh, Yang-Yei Seo, Min A Woo, Safedin Beqaj, and Sungwhan An Jae Sung LimJae Sung Lim More articles by this author , Bo-Ram BangBo-Ram Bang More articles by this author , Justin J. LeeJustin J. Lee More articles by this author , Ki Hak SongKi Hak Song More articles by this author , TaeJeong OhTaeJeong Oh More articles by this author , Yang-Yei SeoYang-Yei Seo More articles by this author , Min A WooMin A Woo More articles by this author , Safedin BeqajSafedin Beqaj More articles by this author , and Sungwhan AnSungwhan An More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002001.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Hematuria patients are evaluated by cystoscopy to confirm the presence of bladder cancer (BCa). However, a large majority of cystoscopies are deemed unnecessary because only 5-20% of hematuria patients are diagnosed with BCa. Urine cytology has suboptimal sensitivity to triage hematuria patients for cystoscopy. As such, the development of a highly sensitive urine-based test for the early detection of BCa in hematuria patients is imperative. Our previous study identified aberrant methylation of the PENK gene (mePENK) as a specific biomarker for BCa and showed that a real-time PCR-based methylation assay for mePENK in urine sediments is a promising solution to early detection of BCa. This study was aimed to demonstrate the clinical validity of urine DNA-based mePENK assay as a useful adjunct to cystoscopy. METHODS: Two independent training and validation studies were conducted. Urine samples were collected from hematuria patients before cystoscopy or surgical treatment. Control urine samples were collected from hematuria patients without BCa. For the PENK methylation assay, two steps of PCR reactions were required: Liner Target Enrichment (LTE) and methylation specific real time PCR (qMSP) coupled with bisulfite conversion. RESULTS: The training set of the clinical study showed that sensitivity of the PENK methylation assay by LTE/qMSP was 91.0% with a specificity of 96.0% in detecting Ta high-grade and all stages of BCa from hematuria patients (Fig. 1). The prospectively designed validation study showed that the urine-based mePENK test demonstrated a sensitivity of 93.0% with a specificity of 92.0% in detecting all of Ta high-grade, Tis, and all stages of BCa from hematuria patients (Fig. 2, interim data). CONCLUSIONS: This study suggests that the urine DNA-based PENK methylation test possesses high potential to be a routine adjunct to cystoscopy in primary diagnostic settings. Use of the PENK methylation test could prevent unnecessary cystoscopy. Source of Funding: This study was sponsored by Genomictree, Inc © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e302-e303 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jae Sung Lim More articles by this author Bo-Ram Bang More articles by this author Justin J. Lee More articles by this author Ki Hak Song More articles by this author TaeJeong Oh More articles by this author Yang-Yei Seo More articles by this author Min A Woo More articles by this author Safedin Beqaj More articles by this author Sungwhan An More articles by this author Expand All Advertisement Loading ...

Validation of Single Nucleotide Variant Assays for Human Leukocyte Antigen Haplotypes HLA-B*15:02 and HLA-A*31:01 Across Diverse Ancestral Backgrounds.

The human leukocyte antigen haplotypes HLA-B*15:02 and HLA-A*31:01 have been linked to life-threatening adverse drug reactions to the anticonvulsants carbamazepine and oxcarbazepine. Identification of these haplotypes via pharmacogenetic techniques facilitates implementation of precision medicine to prevent such reactions. Using reference samples from diverse ancestral origins, we investigated the test analytical validity (i.e., ability to detect whether or not the haplotypes were present or absent) of TaqMan assays for single nucleotide variants previously identified as potentially being able to “tag” these haplotypes. A TaqMan custom assay for rs10484555 and an inventoried assay for rs17179220 and were able to identify with 100% sensitivity and 100% specificity HLA-B*15:02 and HLA-A*31:01 respectively. A custom assay for rs144012689 that takes into account a neighboring single nucleotide variant with manual calling was also able to identify HLA-B*15:02 with 100% sensitivity and 100% specificity. A custom assay for rs1061235 identified HLA-A*31:01 with 100% sensitivity and 95% specificity. The slight reduction in specificity for the latter was owing to another haplotype (HLA-A*33:03) also being detected. While any positive call using the rs1061235 assay could therefore be further investigated, as the presence of the HLA-A*31:01 haplotype confers adverse drug reaction risk, the absence of false negatives (indexed by sensitivity) is more important than false positives. In summary, we present validated TaqMan assay methodology for efficient detection of HLA haplotypes HLA-B*15:02 and HLA-A*31:01. Our data are relevant for other genotyping technologies that identify, or have the potential to identify, these haplotypes using single nucleotide variants.

Analytical Validation of a Pan-Cancer Panel for Cell-Free Assay for the Detection of EGFR Mutations.

Liquid biopsies have increasingly shown clinical utility. Although next-generation sequencing has been widely used for the detection of somatic mutations from plasma, performance characteristics vary by platform. Therefore, thorough validation is mandatory for clinical use. This study aimed to evaluate the analytical validity of the Oncomine Pan-Cancer Cell-Free Assay. A massively parallel sequencing for the assay was performed using the Ion S5 XL System with Ion 540 kit. The analytical sensitivity and precision were evaluated using pre-characterized reference materials. The specificity was evaluated using plasma from healthy subjects. A comparison with the Cobas EGFR Mutation Test v2 was performed using reference materials and plasma from lung cancer patients. For SNVs and short indels, the analytical sensitivities at variant allele frequencies (VAFs) of 0.1%, 0.5%, and 1% were 50%, 93.4%, and 100% with 20 ng of input, respectively. The overall precision of the true positive variants was 98% at a VAF of 1% with 20 ng input. The assay showed a similar sensitivity to that of the Cobas EGFR Mutation Test v2 at a VAF of 0.5% with 20 ng of input and 100% concordance on clinical samples. The Pan-Cancer Cell-Free Assay can be applied to detect EGFR mutations in advanced lung cancer patients, although follow-up studies will be needed to evaluate the analytical validity for other types of genes and aberrations using clinical samples.

Development and Validation of a Simoa Assay for Determination of Recombinant Batroxobin in Human Serum.

Recombinant batroxobin (S3101) is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system. A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies. Therefore, a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101. In this study, a sensitive bioanalytical method was developed and validated, using a Quanterix single molecular array (Simoa) assay. Moreover, to thoroughly assess the platform, enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed, and their performance was compared with that of this novel technology platform. The assay was validated in compliance with the current guidelines. Measurements with the Simoa assay were precise and accurate, presenting a valid assay range from 6.55 to 4000 pg/mL. The intra- and inter-run accuracy and precision were within -19.3% to 15.3% and 5.5% to 17.0%, respectively. S3101 was stable in human serum for 280 days at -20°C and -70°C, for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature (22°C-28°C), respectively, and after five and two freeze-thaw cycles at -70°C and -20°C, respectively. The Simoa assay also demonstrated sufficient dilution linearity, assay sensitivity, and parallelism for quantifying S3101 in human serum. The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.

Age Trends in Growth and Differentiation Factor-11 and Myostatin Levels in Healthy Men, and Differential Response to Testosterone, Measured Using Liquid Chromatography-Tandem Mass Spectrometry.

Growth and differentiation factor (GDF)-11 controls embryonic development and has been proposed as an antiaging factor. GDF-8 (myostatin) inhibits skeletal muscle growth. Difficulties in accurately measuring circulating GDF-11 and GDF-8 have generated controversy. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of circulating GDF-8 and GDF-11 that employs denaturation, reduction, and alkylation; cation-exchange solid-phase extraction; tryptic digestion; followed by separation and quantification using 2 signature peptides for multiple reaction monitoring and C-terminal [13C615N4]-Arg peptides as internal standards. We evaluated age trends in serum GDF-11 and GDF-8 concentrations in community-dwelling healthy men, 19 years or older, and determined the effects of graded testosterone doses on GDF-8 and GDF-11 concentrations in healthy men in a randomized trial. The assay demonstrated linearity over a wide range, lower limit of quantitation 0.5 ng/mL for both proteins, and excellent precision, accuracy, and specificity (no detectable cross-reactivity of GDF-8 in GDF-11 assay or of GDF-11 in GDF-8 assay). Mean ± SD (median ± 1QR) GDF-8 and GDF-11 levels in healthy community-dwelling men, 19 years and older, were 7.2 ± 1.9 (6.8 ± 1.4) ng/mL. Neither GDF-8 nor GDF-11 levels were related to age or body composition. Testosterone treatment significantly increased serum GDF-8 but not GDF-11 levels. The LC-MS/MS method for the simultaneous measurement of circulating total GDF-8 and GDF-11 demonstrates the characteristics of a valid assay. Testosterone treatment increased GDF-8 levels, but not GDF-11. Increase in GDF-8 levels by testosterone treatment, which increased muscle mass, suggests that GDF-8 acts as a chalone to restrain muscle growth.

Validity of different copeptin assays in the differential diagnosis of the polyuria-polydipsia syndrome

The aim of this study was to correlate three commercially available copeptin assays and their diagnostic accuracy in the differential diagnosis of the polyuria-polydipsia syndrome. Analyzed data include repeated copeptin measures of 8 healthy volunteers and 40 patients with polyuria-polydipsia syndrome undergoing osmotic stimulation and of 40 patients hospitalized with pneumonia. Copeptin was measured using the automated Brahms KRYPTOR, the manual Brahms LIA and the manual Cloud Clone ELISA assay. Primary outcome was the interrater correlation coefficient (ICC) and diagnostic accuracy in the polyuria-polydipsia syndrome of the three assays. In healthy volunteers, there was a moderate correlation for the KRYPTOR and LIA (ICC 0.74; 95% CI 0.07 to 0.91), and a poor correlation for the KRYPTOR and ELISA (ICC 0.07; 95% CI − 0.06 to 0.29), as for the LIA and ELISA (ICC 0.04; 95% CI − 0.04 to 0.17). The KRYPTOR had the highest diagnostic accuracy (98% (95% CI 83 to100)), comparable to the LIA (88% (95% CI 74 to 100)), while the ELISA had a poor diagnostic accuracy (55% (95% CI 34 to 68)) in the differential diagnosis of the polyuria-polydipsia syndrome. The KRYPTOR and LIA yield comparable copeptin concentrations and high diagnostic accuracy, while the ELISA correlates poorly with the other two assays and shows a poor diagnostic accuracy for polyuria-polydipsia patients. The current copeptin cut-off is valid for the KRYPTOR and LIA assay. Our results indicate that interpretation with other assays should be performed with caution and separate validation studies are required before their use in differentiating patients with polyuria-polydipsia syndrome.Trial registration: NCT02647736 January 6, 2016/NCT01940614 September 12, 2013/NCT00973154 September 9, 2009.

Validity of Different Copeptin Assays in the Differential Diagnosis of the Polyuria-Polydipsia Syndrome

Background: Copeptin is used in the differential diagnosis of diabetes insipidus. Different copeptin immunoassays exist but inter-assay comparability is unclear. The aim of this study was to correlate three commercially available copeptin assays and their diagnostic accuracy in the differential diagnosis of the polyuria-polydipsia-syndrome. Methods: Analyzed data include three different studies: repeated copeptin measures of 8 healthy volunteers undergoing osmotic stimulation; copeptin measures of 40 patients hospitalized with pneumonia; osmotically stimulated copeptin measures of 40 patients with polyuria-polydipsia-syndrome. Copeptin was measured using the automated B.R.A.H.M.S. KRYPTOR, the manual B.R.A.H.M.S. LIA and the manual Cloud Clone ELISA assay. Primary outcome was the diagnostic accuracy in the polyuria-polydipsia-syndrome. Results: In total, 150 copeptin measurements were analyzed. In healthy volunteers, there was a moderate correlation for the KRYPTOR and LIA (interrater correlation coefficient (ICC) 0.74; 95%-CI 0.07-0.91), and a poor correlation for the KRYPTOR and ELISA (ICC 0.07; 95%-CI -0.06-0.29), as for the LIA and ELISA (ICC 0.04; 95%-CI -0.04-0.17). The KRYPTOR had the highest diagnostic accuracy (98% (95%-CI: 83-100)), comparable to the LIA (88% (95%-CI: 74-100)), while the ELISA had a poor diagnostic accuracy (55% (95%-CI: 34-68)) in the differential diagnosis of the polyuria-polydipsia-syndrome. Conclusion: The KRYPTOR and LIA yield comparable copeptin levels and a high diagnostic accuracy, while the ELISA correlates poorly with the other two assays and shows a poor diagnostic accuracy between polyuria-polydipsia patients. Redefining cut-off levels for copeptin assays other than KRYPTOR and LIA must take place before their use in the differential diagnosis of diabetes insipidus.

Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults

BackgroundVolunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials.MethodsA previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed.ResultsThe reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples.ConclusionsThe 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

Predictive biomarkers and clinical evidence.

Predictive biomarkers play an important role in our efforts to individualize pharmacotherapy, and within recent years, a number of different types of assays have been introduced. These biomarkers may potentially support the selection and dosage of specific drugs in order to maximize efficacy and minimize adverse reactions in the individual patient. However, in many instances, the scientific and clinical evidence is insufficient to support the prescribing decision. When predictive biomarkers are used to guide pharmacotherapy, it is important to secure that decisions are based on solid clinical evidence. Here, the regulatory authorities, especially the FDA, have been at the forefront in relation to regulate this type of biomarker assay in order to secure patient safety. The approval process for companion diagnostics is an example of this effort, where the scientific validity of the biomarker and assay is in focus. With the approaching implementation of the new IVD Regulation, greater attention will also be paid to analytical and clinical validity of biomarker assays in the EU. For any type of predictive biomarker assay, including pharmacogenetic and tumour profiling tests, the clinical evidence needs to be in place before they are used routinely in the clinic.

Validity of Urinary Monoamine Assay Sales Under the "Spot Baseline Urinary Neurotransmitter Testing Marketing Model" [Retraction

[This retracts the article DOI: 10.2147/IJNRD.S22783.].

A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule

Classic antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.

PD-L1 expression in paired biopsies and surgical specimens in gastric adenocarcinoma: A digital image analysis study

Programmed death-ligand 1 (PD-L1) expression in biopsies of gastric carcinoma may predict the results in corresponding surgical specimens. We compared PD-L1 immunohistochemistry (IHC) 22C3 pharmDx expression in paired biopsy and resection specimens. We also characterized the validity of a new PD-L1 assay using digital image analysis.PD-L1 IHC with 22C3 pharmDx and clone 73−10 was performed in 224 gastric cancer tissues (112 biopsies and paired surgical tissues) and the specimens were analyzed with the Leica Aperio Imagescope. For statistical analyses, the area under the receiver operating characteristic curve and R package were used.With 22C3 pharmDx, a PD-L1 combined positive score of ≥1 was found in 36 biopsied (32.14 %) and 53 surgical (47.32 %) samples. PD-L1 expression results were concordant in 71 cases (63.4 %) and discordant in 41 (36.6 %). The overall discordance rate was 36.61 % (95 % confidence interval 2.101–8.983) and the κ value was 0.254 with fair agreement. The sensitivity and specificity of biopsy PD-L1 to predict the results of the surgical specimen was 62 % and 73 %, respectively. The correlation of 22C3 pharmDx and clone 73−10 was high (correlation coefficient = 0.88). When only tumor cell staining was compared, this correlation was increased (correlation coefficient = 0.95).Our results indicated moderate association of PD-L1 expression between gastric biopsies and corresponding resected tumors. Results of PD-L1 assay with 73−10 are comparable to 22C3 pharmDx results.