Intra-assay Coefficient Of Variation Research Articles

The reliability, sensitivity and specificity of fluorescent oxidation products for measuring global oxidative stress in vivo

Introduction To examine the reliability, sensitivity, and specificity of fluorescent oxidation products (FlOPs; markers of global oxidative damage) for measuring global oxidative stress. Methods To improve FlOP measurement reliability, we mixed plasma samples with the extractant at eight ratios and measured FlOPs with a fluorescent microplate reader (excitation/emission wavelengths 320/420 nm denoted FlOP_320; 360/420 nm [FlOP_360]; and 400/475 nm [FlOP_400]). In a human study, we examined the reliability of an improved FlOP measurement. In an animal study, we examined the sensitivity and specificity of FlOPs for measuring D-galactose induced global oxidative stress. Results At a 1:20 (plasma/extractant) mixture ratio, the overall inter-/intra-assay coefficients of variation (CV) for FlOP measurements among healthy and coronary heart disease participants were <3.6%/<2.7% and <4.4%/<2.0%, respectively. On day 30 of the animal experiment, the Pearson correlations (r) of FlOP_320 and FlOP_360 with D-galactose dose were 0.816 and 0.801, respectively, which were 3-12 times higher than those of malondialdehyde (0.183), 8-hydroxyguanosine (0.157), pentosidine (0.254), and nitrotyrosine (0.068) for measuring D-galactose-induced oxidative damage. FlOP_360 (r = 0.225, P = 0.045), but not FlOP_320 and FlOP_400, were significantly correlated with c-reactive protein, a marker of inflammation. Conclusions FlOP_320 are reliable, sensitive and specific markers for measuring global oxidative stress. Key policy highlights Fluorescent oxidation products (FlOPs) are a reliable marker of oxidative stress FlOPs at 320/420 nm are more sensitive than traditional markers FlOP at 320/420 nm did not reflect inflammation as quantified by c-reactive protein

First Report on the Molecular Detection of Canine Astrovirus (CaAstV) in Dogs with Gastrointestinal Disease in Ecuador Using a Fast and Sensitive RT-qPCR Assay Based on SYBR Green®.

Enteric viruses are responsible for a significant number of gastrointestinal illnesses in dogs globally. One of the main enteric viruses is the canine astrovirus (CaAstV), which causes diarrhea in dogs of various ages. It is linked to symptoms such as diarrhea, vomiting, depression and a significant mortality rate due to gastrointestinal disorders. It is a single-stranded positive RNA virus, with three open reading frames, ORF1a, ORF1b and ORF2, where the last one codes for the virus capsid protein and is the most variable and antigenic region of the virus. The aim of this work is to develop and standardize a quick detection method to enable the diagnosis of this etiological agent in dogs with gastroenteritis in Ecuador in order to provide prompt and suitable treatment. The assay was specific for amplification of the genome of CaAstV, as no amplification was shown for other canine enteric viruses (CPV-2, CCoV and CDV), sensitive by being able to detect up to one copy of viral genetic material, and repeatable with inter- and intra-assay coefficients of variation of less than 10% between assays. The standard curve showed an efficiency of 103.9%. For the validation of this method, 221 fecal samples from dogs affected with gastroenteritis of various ages from different provinces of Ecuador were used. From the RT-qPCR protocol, 119 samples were found positive for CaAstV, equivalent to 53.8% of the samples processed. CaAstV was detected in dogs where both the highest virus prevalence in the tested strains and the highest viral loads were seen in the younger canine groups up to 48 weeks; in addition, different strains of the virus were identified based on a sequenced fragment of ORF1b, demonstrating the first report of the presence of CaAstV circulating in the domestic canine population affected by gastroenteritis in Ecuador, which could be associated with the etiology and severity of enteric disease.

Serum anti-Müllerian hormone is an indirect predictor of ovarian reserve in domestic cats

Anti-Müllerian hormone (AMH) serves as an indirect marker for predicting primordial follicles that are representative of ovarian reserve. In this study the possibility of using AMH and age to predict the ovarian reserve in domestic cats. Ovaries and blood were collected from 30 cats undergoing routine ovariohysterectomy. The animals were divided into three age groups: prepubertal (<4 mo, n = 10), adult (1–5 y, n = 10), and senior (>5 y, n = 10). Blood was collected at surgery for serum AMH measurements using the AMH Gen II ELISA kit. The intra-assay coefficient of variation (CV) and inter-assay CV were 3.56 % and 7.68 %, respectively. One side of the ovary was processed to determine AMH localization using immunohistochemistry and for a histological count of follicles, which is the gold standard. The expression of AMH protein was quantified from the contralateral ovary by Western blot analysis. Primordial follicles exhibited the most pronounced inverse relationship with age (rho = −0.779, P < 0.05), followed by a positive association with serum AMH concentration (rho = 0.490, P < 0.05), indicating that both age and AMH are potential markers indicative of primordial follicles. Furthermore, secondary (rho = 0.651, P < 0.05) and small antral follicles (rho = 0.648, P < 0.05) were identified as the major sources of circulating AMH, as indicated by the stronger correlation with serum AMH concentrations compared with primary follicles. However, there was no significant correlation between the expression of AMH protein and other factors, including age, primordial follicles, primary follicles, secondary follicles, small antral follicles, and serum AMH concentration. A model for predicting primordial follicle number using serum AMH concentration (AIC = 672.66, P < 0.05) and age (AIC = 668.93, P < 0.05) was established. In conclusion, both serum AMH concentration and age may serve as comparable markers of ovarian reserve in domestic cats. Moreover, AMH is particularly useful in situations where age information is not available.

Development and validation of an assay to quantify plasma cell-free human papillomavirus DNA for 13 high-risk types that cause 98% of HPV-positive cancers.

6065 Background: Plasma cell-free HPV DNA (cfHPV) may be a valuable tool for identification of patients with local-regionally advanced HPV-positive cancers at risk for recurrence after chemoradiation. Technical validation is required for use as an integral biomarker in a prospective clinical trial. Methods: Cell-Free 13 (i.e., CF13) is a digital-droplet PCR assay for quantification of 13 high-risk HPV types and control ERV3 in plasma that considered both variant sequences into primer/probe design and a distance matrix in phylogenetic analysis in multiplex combinations. Oligonucleotides/plasmids encoding type-specific E6/E7 regions were used as positive controls. Limit of blank, detection, quantification (LoB, LoD, LoQ), linear range, inter- and intra-assay coefficients of variation (CoV), and type-specific cross-reactivity were determined as were sensitivity and specificity for an HPV-positive diagnosis in a cohort of 278 oropharyngeal/unknown primary/oral cavity cancers tested for HPV by E6/E7 mRNA qRT-PCR or p16 IHC and HPV in situ hybridization. Results: Under identical assay conditions, the LoB, LoD and linear range were &lt;1, 5 and 5 to 200,000 virus copies without HPV type-specific cross-reactivity. Multiplexing had no effect on LoD or linearity. LoQ was 16 copies/ml plasma for all 13 HPV types. For 80 and 10,000 copies per ml, inter-assay CoV ranged from ~15-28% and ~3.2-4.6% and intra-assay CoV ranged from ~16-38% and 0.2-3.3%, respectively. cfDNA purification method (manual v automated; extraction efficiency 142% and 91% for 16 and 10,000 HPV16 copies/ml), input plasma volume (1, 2, or 4 ml), total background cfDNA (&lt;1800ng) or genomic DNA (&lt;700ng) did not affect quantification. For a diagnosis of HPV-positive cancer, sensitivity and specificity were 91.1% (214/235) and 97.7% (42/43), respectively. cfHPV was associated with gender, race, T stage, N stage, and primary treatment modality. When compared to below the median (≤230 copies/ml), cfHPV above the median was associated with worse progression-free survival (HR=2.26, 95% CI 1.23-4.17, p=0.009) in univariate analysis. However, this was no longer significant after adjustment for age, T stage, and M stage (HRadj=1.78, 95% CI 0.95-3.35, p=0.07). Conclusions: CF13 has high sensitivity, accuracy, precision, linearity, HPV type-specificity, and robustness, offering a rigorously validated assay applicable to ~98% of patients with HPV-positive cancer. CF13 had excellent clinical sensitivity and specificity for a diagnosis of HPV-positive OPC.

A qPCR Assay for the Quantification of Selected Genotypic Variants of Spodoptera frugiperda Multiple Nucleopolyhedrovirus (Baculoviridae).

Alphabaculoviruses are lethal dsDNA viruses of Lepidoptera that have high genetic diversity and are transmitted in aggregates within proteinaceous occlusion bodies. This mode of transmission has implications for their efficacy as biological insecticides. A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV-NIC) comprising nine genotypic variants has been the subject of considerable study due to the influence of variant interactions on the insecticidal properties of mixed-variant occlusion bodies. As part of a systematic study on the replication and transmission of variant mixtures, a tool for the accurate quantification of a selection of genotypic variants was developed based on the quantitative PCR technique (qPCR). First, primer pairs were designed around a region of high variability in four variants named SfNic-A, SfNic-B, SfNic-C and SfNic-E to produce amplicons of 103-150 bp. Then, using cloned purified amplicons as standards, amplification was demonstrated over a dynamic range of 108-101 copies of each target. The assay was efficient (mean ± SD: 98.5 ± 0.8%), reproducible, as shown by low inter- and intra-assay coefficients of variation (<5%), and specific to the target variants (99.7-100% specificity across variants). The quantification method was validated on mixtures of genotype-specific amplicons and demonstrated accurate quantification. Finally, mixtures of the four variants were quantified based on mixtures of budded virions and mixtures of DNA extracted from occlusion-derived virions. In both cases, mixed-variant preparations compared favorably to total viral genome numbers by quantification of the polyhedrin (polh) gene that is present in all variants. This technique should prove invaluable in elucidating the influence of variant diversity on the transmission and insecticidal characteristics of this pathogen.

Quantification of multiple steroid hormones in serum and human breast cancer tissue by liquid chromatography-tandem mass spectrometry analysis.

Systemic and local steroid hormone levels may function as novel prognostic and predictive biomarkers in breast cancer patients. We aimed at developing a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of multiple, biologically pivotal steroid hormones in human serum and breast cancer tissue. The quantitative method consisted of liquid-liquid extraction, Sephadex LH-20 chromatography for tissue extracts, and analysis of steroid hormones by liquid-chromatography-tandem mass spectrometry. We analyzed serum and tissue steroid hormone levels in 16 and 40 breast cancer patients, respectively, and assessed their correlations with clinical parameters. The method included quantification of nine steroid hormones in serum [including cortisol, cortisone, corticosterone, estrone (E1), 17β-estradiol (E2), 17α-hydroxyprogesterone, androstenedione (A4), testosterone and progesterone) and six (including cortisone, corticosterone, E1, E2, A4, and testosterone) in cancer tissue. The lower limits of quantification were between 0.003-10 ng/ml for serum (250 µl) and 0.038-125 pg/mg for tissue (20 mg), respectively. Accuracy was between 98%-126%, intra-assay coefficient of variations (CV) was below 15%, and inter-assay CV were below 11%. The analytical recoveries for tissue were between 76%-110%. Tissue levels of E1 were positively correlated with tissue E2 levels (p<0.001), and with serum levels of E1, E2 and A4 (p<0.01). Tissue E2 levels were positively associated with serum E1 levels (p=0.02), but not with serum E2 levels (p=0.12). The levels of tissue E2 and ratios of E1 to A4 levels (an index for aromatase activity) were significantly higher in patients with larger tumors (p=0.03 and p=0.02, respectively). The method was convenient and suitable for a specific and accurate profiling of clinically important steroid hormones in serum. However, the sensitivity of the profile method in steroid analysis in tissue samples is limited, but it can be used for the analysis of steroids in breast cancer tissues if the size of the sample or its steroid content is sufficient.

A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide.We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap anddeveloped a multiplex crystal digital PCR (cdPCR) method after optimizing the primerconcentration, probe concentration, and annealing temperature.The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus.

Feline coronavirus (FCoV), feline panleukopenia virus (FPV), and feline leukemia virus (FeLV) are prevalent throughout China and significantly threaten cat health. These viruses cause similar manifestations and pathological damage. Rapid and accurate diagnosis depends on detection in the laboratory. This study aimed to establish a reliable and rapid method for accurate detection of FCoV, FPV, and FeLV so that a definite diagnosis can be made and effective measures can be taken to prevent and control viral infection. We designed three pairs of specific primers and probes for the detection of FCoV 5' untranslated region, FPV viral protein 2, and FeLV pol genes. Recombinant plasmid constructs were generated for use as standard plasmid constructs. Optimal reaction conditions, including primer and probe concentrations, reaction cycles, and annealing temperatures, were obtained on the basis of optimization tests. One-step triplex real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was successfully established to simultaneously detect FCoV, FPV, and FeLV. The specificity, sensitivity, and repeatability of the assay were analyzed, and its applicability was validated by testing 1175 clinical samples. One-step triplex RT-qPCR had a high degree of specificity only for the detection of FCoV, FPV, and FeLV; it had high sensitivity with limits of detection of 139.904, 143.099, and 152.079 copies/reaction for p-FCoV, p-FPV, and p-FeLV standard plasmid constructs, respectively, and it had reliable repeatability with 0.06%-0.87% intra-assay coefficients of variations. A total of 1175 clinical samples were examined for FCoV, FPV, and FeLV using triplex RT-qPCR, and the FCoV, FPV, and FeLV positivity rates were 18.47%, 19.91%, and 47.57%, respectively. The clinical sensitivity and specificity of one-step triplex RT-qPCR were 93.07% and 97.99%, respectively. We developed a rapid and reliable one-step triplex RT-qPCR method for the detection of FCoV, FPV, and FeLV, which could be used as a diagnostic tool for clinical monitoring and diagnosis.

Simultaneous Detection of Porcine Respiratory Coronavirus, Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus, and Pseudorabies Virus via Quadruplex One-Step RT-qPCR.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.

Advancing cortisol measurements in zebrafish: Analytical validation of a commercial ELISA kit for skin mucus cortisol analysis

Cortisol is the main stress biomarker used for zebrafish. However, zebrafish small size made it challenging to extract cortisol without harming or killing the fish. Thus, researchers adopted a terminal method, the trunk cortisol, as standard practice. Here, we developed and validated an alternative and minimally invasive technique for measuring cortisol in the skin mucus of adult zebrafish, using a commercial enzyme-linked immunosorbent assay (ELISA). For this, AB zebrafish were randomly assigned to a precision, accuracy, and specificity test. Each sample contained the skin mucus of five to ten fish or one fish trunk. The cortisol was extracted using methanol as organic solvent. The results obtained showed an adequate precision (intra-assay coefficient of variation (CV) <15%; inter-assay CV = 26%), accuracy (CV <120%), and specificity (r2 =0.96–0.98) for skin mucus cortisol levels, as well as for trunk cortisol.•A commercial ELISA was analytically validated to measure cortisol in the skin mucus of zebrafish.•Skin mucus cortisol is a non-terminal method that reduce the number of animals used and allows longitudinal studies.

The development, evaluation, performance, and validation of micro-PCR and extractor for the quantification of HIV-1 &-2 RNA

HIV infection has been a global public health threat and overall reported ~ 40 million deaths. Acquired immunodeficiency syndrome (AIDS) is attributed to the retroviruses (HIV-1/2), disseminated through various body fluids. The temporal progression of AIDS is in context to the rate of HIV-1 infection, which is twice as protracted in HIV-2 transmission. Q-PCR is the only available method that requires a well-developed lab infrastructure and trained personnel. Micro-PCR, a portable Q-PCR device, was developed by Bigtec Labs, Bangalore, India. It is simple, accurate, fast, and operationalised in remote places where diagnostic services are inaccessible in developing countries. This novel micro-PCR determines HIV-1 and HIV-2 viral load using a TruePrep™ extractor device for RNA isolation. Five ml blood samples were collected at the blood collection centre at AIIMS, New Delhi, India. Samples were screened for serology, and a comparison of HIV-1/2 RNA was done between qPCR and micro-PCR in the samples. The micro-PCR assay of HIV-RNA has compared well with those from real-time PCR (r = 0.99, i < 0.002). Micro-PCR has good inter and intra-assay reproducibility over a wide dynamic range (1.0 × 102–1.0 × 108 IU/ml). The linear dynamic range was 102–108 IU/ml. The clinical and analytical specificity of the assay was comparable, i.e., 100%. Intra-assay and inter-assay coefficients of variation ranged from 1.17% to 3.15% and from 0.02% to 0.46%, respectively. Moreover, due to the robust, simple, and empirical method, the Probit analysis has also been done for qPCR LODs to avoid uncertainties in target recoveries. The micro-PCR is reliable, accurate, and reproducible for early detection of HIV-1 and HIV-2 viral loads simultaneously. Thus, it can easily be used in the field and in remote places where quantification of both HIV-1/2 is not reachable.

A triplex crystal digital PCR for the detection of genotypes I and II African swine fever virus.

African swine fever (ASF) is a highly contagious and lethal viral disease that causes severe hemorrhagic fever in pigs. It keeps spreading around the world, posing a severe socioeconomic risk and endangering biodiversity and domestic food security. ASF first outbroke in China in 2018, and has spread to most provinces nationwide. Genotypes I and II ASF virus (ASFV) as the etiological pathogens have been found in China. In this study, three pairs of specific primers and probes targeting the ASFV B646L gene, F1055L gene, and E183L gene were designed to detect universal, genotype I, and genotype II strains, respectively. A triplex crystal digital PCR (cdPCR) was established on the basis of optimizing various reaction conditions. The assay demonstrated remarkably sensitive with low limits of detection (LODs) of 5.120, 4.218, 4.588 copies/reaction for B646L, F1055L, and E183L gene, respectively; excellent repeatability with 1.24-2.01% intra-assay coefficients of variation (CVs) and 1.32-2.53% inter-assay CVs; good specificity for only detection of genotypes I and II ASFV, without cross-reactivity with PCV2, PRV, SIV, PRRSV, PEDV, FMDV, and CSFV. The triplex cdPCR was used to test 1,275 clinical samples from Guangxi province of China, and the positivity rates were 5.05, 3.22, and 1.02% for genotype I, genotype II, and co-infection of genotypes I and II, respectively. These 1,275 clinical samples were also detected using a reported reference triplex real-time quantitative PCR (qPCR), and the agreements of detection results between these two methods were more than 98.98%. In conclusion, the developed triplex cdPCR could be used as a rapid, sensitive, and accurate method to detect and differentiate genotypes I and II strains of ASFV.

Measurement of Feline Alpha-1 Acid Glycoprotein in Serum and Effusion Using an ELISA Method: Analytical Validation and Diagnostic Role for Feline Infectious Peritonitis.

Alpha-1 acid glycoprotein (AGP) may support a clinical diagnosis of feline infectious peritonitis (FIP). In this study, we assessed the analytical and diagnostic performances of a novel ELISA method to measure feline AGP. AGP was measured in sera and effusions from cats with FIP (n = 20) or with other diseases (n = 15). Precision was calculated based on the coefficient of variation (CV) of repeated testing, and accuracy was calculated by linearity under dilution (LUD). The test is precise (intra-assay CVs: <6.0% in individual samples, <15.0% in pooled samples; inter-assay CVs <11.0% and <15.0%) and accurate (serum LUD r2: 0.995; effusion LUD r2: 0.950) in serum and in effusions. AGP is higher in cats with FIP than in other cats in both serum (median: 1968, I-III interquartile range: 1216-3371 μg/mL and 296, 246-1963 μg/mL; p = 0.009) and effusion (1717, 1011-2379 μg/mL and 233, 165-566 μg/mL; p < 0.001). AGP discriminates FIP from other diseases (area under the receiver operating characteristic curve: serum, 0.760; effusion, 0.877), and its likelihood ratio is high (serum: 8.50 if AGP > 1590 μg/mL; effusion: 3.75 if AGP > 3780 μg/mL). This ELISA method is precise and accurate. AGP in serum and in effusions is a useful diagnostic marker for FIP.

An immunochromatographic strip sensor for marbofloxacin residues.

Marbofloxacin (MBF) was once widely used as a veterinary drug to control diseases in animals. MBF residues in animal food endanger human health. In the present study, an immunochromatographic strip assay (ICSA) utilizing a competitive principle was developed to rapidly detect MBF in beef samples. The 50% inhibitory concentration (IC50) and the limit of detection (LOD) of the ICSAs were 2.5 ng/mL and 0.5 ng/mL, respectively. The cross-reactivity (CR) of the MBF ICSAs to Ofloxacin (OFL), enrofloxacin (ENR), norfloxacin (NOR), and Ciprofloxacin (CIP) were 60.98%, 32.05%, 22.94%, and 23.58%, respectively. The CR for difloxacin (DIF) and sarafloxacin (SAR) was less than 0.1%. The recovery rates of MBF in spiked beef samples ranged from 82.0% to 90.4%. The intra-assay and interassay coefficients of variation (CVs) were below 10%. In addition, when the same authentic beef samples were detected in a side-by-side comparison between the ICSAs and HPLC‒MS, no statistically significant difference was observed. Therefore, the proposed ICSAs can be a useful tool for monitoring MBF residues in beef samples in a qualitative and quantitative manner.

Abstract 81: High-resolution monitoring of B cell isotype switching for biomarker analysis of adenosine pathway inhibition immunotherapy

Abstract Background B cell isotype switching gives rise to IgG-, IgA-, and IgE-expressing B cells and is an essential step in generating effective humoral responses to antigen challenge. Within the tumor microenvironment, extracellular adenosine may contribute to cancer immune evasion by inhibiting isotype switching through interaction with A2aR, a possibility that has driven investigation of adenosine pathway inhibition as an immunotherapeutic modality. In this context, methods to quantify isotype switching may provide insight into the pharmacodynamics of candidate agent activity and serve as a source of predictive biomarkers of response. We recently clinically validated the Oncomine IGH-LR assay for the measurement of CLL somatic hypermutation (SHM). Given that this assay also detects isotype switching events, we retrospectively analyzed our validation dataset to determine the suitability of the assay as a potential tool for biomarker analysis of adenosine pathway inhibition immunotherapy. Methods The Oncomine IGH-LR assay determines B cell SHM and isotype through targeted next-generation sequencing of IGH chains from RNA. The assay software organizes detected IGH chains into B cell clonal lineages, of which a subset may comprise multi-isotype lineages indicative of isotype switching events. The software further reports secondary repertoire features such as clonality/evenness. Total RNA was extracted from peripheral blood mononuclear cells or bone marrow aspirates from patients identified as having &amp;gt;10% monoclonal B-cell population by flow cytometry. Intra-assay precision was evaluated by processing RNA from 7 samples in triplicate and sequencing in the same run. Inter-assay precision was evaluated by having different operators process 10 samples on different days with different sequencing barcodes. Dominant clone isotype and clonal lineage assignment, total isotype abundance, and repertoire clonality were evaluated across replicates. Results Overall, 100% concordance was achieved for dominant clone isotype and clonal lineage calls for both intra- and inter-assay studies. Intra-assay and inter-assay median coefficient of variation (CV) for reported isotype frequencies ranged from 0.10-0.16 and 0.15-0.28, respectively. Intra-assay and inter-assay median CV for clonality (1 - Normalized Shannon Entropy) was 0.06 and 0.04, respectively. Conclusions We observe high reproducibility and repeatability of the IGH-LR assay for B cell isotyping, clonal lineage analysis, and clonality assessment. Multi-isotype clonal lineages are automatically reported by the software and provide a means to directly monitor isotype switching events in the context of antigen challenge or immunomodulation. Taken together, we anticipate this assay to be a useful tool for biomarker analysis of adenosine pathway inhibition immunotherapy. Citation Format: Ramadevi Prathapam, Kristen Champion, Anne Burkhardt, Chelsey L. Smith, Rajesh Singh, Timothy J. Looney. High-resolution monitoring of B cell isotype switching for biomarker analysis of adenosine pathway inhibition immunotherapy [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 81.

First Specific Detection of Mammalian Orthoreovirus from Goats Using TaqMan Real-Time RT-PCR Technology.

Mammalian orthoreovirus (MRV) infections are ubiquitous in multiple mammalian species including humans, and mainly causes gastroenteritis and respiratory disease. In this study, we developed a rapid and sensitive TaqMan qRT-PCR method for MRV detection based on the primers and probe designed within the conserved L1 gene. The qRT-PCR assay was evaluated for its sensitivity, specificity, efficiency and reproducibility. It was found that the detection sensitivity was equivalent to 10 DNA copies/μL, and the standard curves had a linear correlation of R2 = 0.998 with an amplification efficiency of 99.6%. The inter- and intra-assay coefficients of variation (CV%) were in the range of 0.29% to 2.16% and 1.60% to 3.60%, respectively. The primer sets specifically amplified their respective MRV segments and had the highest detection sensitivities of 100.25 TCID50/mL with amplification efficiencies of 99.5% (R2 = 0.999). qRT-PCR was used for MRV detection from samples of sheep, goats, and calves from four regions in China, and the overall MRV prevalence was 8.2% (35/429), whereas 17/429 (4.0%) were detected by RT-PCR and 14/429 (3.3%) by virus isolation. The qRT-PCR assay showed significantly higher sensitivity than RT-PCR and virus isolation. Results from an epidemiological survey indicated that the positive rate of MRV in rectal swabs from sheep and goats tested in Shaanxi, Jiangsu, and Xinjiang were 9/80 (11.3%), 12/93 (12.9%) and 14/128 (10.9%), respectively. In goats and sheep, MRV prevalence was obviously associated with season and age, with a high positive rate of more than 8% during September to April and approximately 13% in small ruminant animals under two months of age. This is the first instance of MRV infection in sheep and goats in China, thus broadening our knowledge of MRV hosts. Consequently, primer optimization for qRT-PCR should not only prioritize amplification efficiency and specificity, but also sensitivity. This assay will contribute to more accurate and rapid MRV monitoring by epidemiological investigation, viral load, and vaccination efficacy.

Added value of a connected glucose meter for glycorrhachia assessment

ObjectivesThe aim of this study was to demonstrate the performance and added value of rapid glucose determination in cerebrospinal fluid using a connected glucometer. Design and MethodsIntra-assay and inter-assay accuracies were calculated using residual clinical samples. Accuracies were measured by comparing the results obtained with the glucometer to those from the central laboratory on a large routine chemistry platform. ResultsThe intra-assay coefficients of variation were between 6.1% and 6.2% for low values (18 mg/dL) and between 5.6% and 6.8% for high values (58 mg/dL). The inter-assay coefficients of variation were between 9.4% and 16.3% for the low values (18 mg/dL) and between 5.7% and 8.7% for the high values (pool; ±75 mg/dL). The regression equation by comparison to the central laboratory was y = 4.08 + 0.82 x, with a coefficient of determination (r2) of 0.95. ConclusionsThe measurement of glycorrhachia with a connected glucometer before the analysis in the central laboratory allows a rapid orientation in the deferential diagnosis of a meningitis of viral vs bacterial origin. The response time is fast (6 s) and requires only a small amount of fluid (1.2 μL), which is important in infants, especially since lumbar puncture is an integral part of the investigation of the origin of a fever in this population.

Evaluation of a commercial proAKAP4 kit for the assessment of fresh and frozen-thawed feline spermatozoa.

ProAKAP4 is gaining increasing attention as a potential marker of semen quality in many species, but while there is a commercial kit for assessing proAKAP4 in the domestic cat, there are no publications about its use. The aim of this study was to evaluate the commercial proAKAP4 kit - Cat 4MID® Kit (SPQI - 4BioDx, Lille, France) for the assessment of feline semen. Semen was collected from 54 male cats by urethral catheterization. After a basic semen evaluation (subjective motility, CASA, viability, morphology), proAKAP4 levels in each sample were assessed using the Cat 4MID® Kit according to the manufacturer's protocol or with some modifications related to incubation time, sample storage conditions and number of spermatozoa used. Finally, the Spearman correlation of proAKAP4 concentration and sperm motility parameters was calculated. The most reliable results (acceptable intraassay coefficient of variation) were obtained with an optimized protocol of overnight incubation and isolation of proAKAP4 protein from 1 × 106 spermatozoa stored at -80°C. For fresh semen, there were no significant correlations between proAKAP4 concentration and sperm motility parameters, despite a strong correlation between motility parameters and sperm viability and morphology. A predominant effect of other sperm parameters and highly variable performance of lysis buffer question the usefulness of Cat 4MID® Kit for the assessment of feline semen. For frozen-thawed semen, there was a moderate, negative correlation between proAKAP4 concentration and two CASA parameters, VAP and VSL. As there were no correlations between proAKAP4 concentration in fresh semen and motility parameters in cryopreserved samples, proAKAP4 cannot be used as freezability marker in cats. More studies are needed to establish potential correlation with long-lasting motility.

Reliability, stability during long-term storage, and intra-individual variation of circulating levels of osteopontin, osteoprotegerin, vascular endothelial growth factor-A, and interleukin-17A

BackgroundStudies in many populations have reported associations between circulating cytokine levels and various physiological or pathological conditions. However, the reliability of cytokine measurements in population studies, which measure cytokines in multiple assays over a prolonged period, has not been adequately examined; nor has stability during sample storage or intra-individual variation been assessed.MethodsWe assessed (1) analytical reliability in short- and long-term repeated measurements; (2) stability and analytical reliability during long-term sample storage, and (3) variability within individuals over seasons, of four cytokines—osteopontin (OPN), osteoprotegerin (OPG), vascular endothelial growth factor-A (VEGF-A), and interleukin-17A (IL-17A). Measurements in plasma or serum samples were made with commercial kits according to standard procedures. Estimation was performed by fitting a random or mixed effects linear model on the log scale.ResultsIn repeated assays over a short period, OPN, OPG, and VEGF-A had acceptable reliability, with intra- and inter-assay coefficients of variation (CV) less than 0.11. Reliability of IL-17A was poor, with inter- and intra-assay CV 0.85 and 0.43, respectively. During long-term storage, OPG significantly decayed (− 33% per year; 95% confidence interval [− 54, − 3.7]), but not OPN or VEGF-A (− 0.3% or − 6.3% per year, respectively). Intra- and inter-assay CV over a long period were comparable to that in a short period except for a slight increase in inter-assay CV of VEGF-A. Within-individual variation was small for OPN and VEGF-A, with intra-class correlations (ICC) 0.68 and 0.83, respectively, but large for OPG (ICC 0.11).ConclusionsWe conclude that OPN and VEGF-A can be reliably measured in a large population, that IL-17A is suitable only for small experiments, and that OPG should be assessed with caution due to degradation during storage and intra-individual variation. The overall results of our study illustrate the need for validation under relevant conditions when measuring circulating cytokines in population studies.

Simultaneous Detection of Prolactin and Growth Hormone Using a Dual-label Time-resolved Fluorescence Immunoassay.

Prolactin (PRL) and growth hormone (GH) are two important hormones secreted by the pituitary gland, and their abnormal levels are often related to disease status. This study aimed to establish a new dual-label time-resolved fluorescence immunoassay (TRFIA) to quantitatively measure PRL and GH levels in serum. A sandwich TRFIA was optimized and established: anti-PRL/GH antibodies immobilized on 96-well plates captured PRL/GH and then banded together with anti-PRL/GH paired antibodies labeled with europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally, a time-resolved analyzer measured the Eu3+/Sm3+ fluorescence values. Clinical serum samples were used to evaluate the detection performance of this method. The sensitivities of this dual-label TRFIA were 0.35 ng/mL and 0.45 ng/mL, respectively, and the detection range was between 0.1 and 1000 ng/mL. All the cross-reactivities were lower than 1.07%. The intra-assay and interassay coefficients of variation were 2.18-7.85% and 2.25-7.30%, respectively. Compared with the registered TRFIA kits, a high Pearson coefficient (r = 0.9626 and 0.9675) was observed. This dual-label TRFIA has high sensitivity, accuracy and specificity with good clinical detection performance, representing a suitable alternative to existing methods for determining PRL and GH levels, and is expected to be used in the clinic in the future.