Serum Aliquots Research Articles

Effect of refrigeration, room temperature, and processing time on serum immunofluorescent antibody titers for Sarcocystis neurona.

Evaluating antibody titers for Sarcocystis neurona for the diagnosis of equine protozoal myeloencephalitis from serum samples is a common practice. However, ensuring timely and proper refrigeration is not always possible. To evaluate immunofluorescent antibody (IFA) titers for S. neurona from serum samples stored at room temperature and 4°C. Twenty-two serum samples. Prospective longitudinal study. Two serum aliquots of 1 mL each were stored at room temperature (20-23.3°C) and 4°C. The unrefrigerated aliquot was immediately tested for IFA titers. Both aliquots were retested on Days 5 and 10 after collection. A paired t test was used to compare IFA titers at different time points. There was no significant difference between IFA titers from baseline with those stored at room temperature at Days 5 (P = .741, 95% CI [-56.83, 78.65]), 10 (P = .677, 95% CI [-50.01, 75.46]), and between 5 and 10 days (P = 0.949, 95% CI [-57.50, 61.14]). There was no significant difference from baseline with those stored at 4°C for Days 5 (P = .964, 95% CI [-81.81, 85.45]), 10 (P = 0.573, 95% CI [-109.4, 62.15]), and between 5 and 10 days (P = .5, 95% CI [-102.6, 51.67]). There was no statistical difference between samples stored at room temperature and 4°C (P = .688, CI [-55.51, 37.33]) on Days 5 and 10 (P = .104, CI [-80.8, 8.07]). Immunofluorescent antibody test titers for S. neurona are stable for up to 10 days at room temperature and 4°C.

Exosomal Micro RNA Isolation in White-Tailed Deer (Odocoileus virginianus) for Diagnostic Biomarker Discovery.

Molecular approaches are becoming more prevalent for the diagnosis of neurodegenerative diseases in human medicine and can be extended to diagnosis of wildlife diseases such as chronic wasting disease and other prion diseases. These diseases have been associated with exosome-bound molecular biomarkers of disease progression, such as proteins and micro RNA molecules (miRNA). We tested and optimized a method for exosomal miRNA isolation from minimally invasive, small-volume serum samples obtained from white-tailed deer (Odocoileus virginianus). We confirmed the isolation of exosomes and optimized a commercially available benchtop kit to obtain sufficient and pure RNA for miRNA sequencing. The selected method for RNA extraction combines two 500-μL serum aliquots into one elution column and re-eluting the final product of the column. We identified 137 miRNA present in healthy white-tailed deer that can be used as a baseline to identify putative miRNA biomarkers of disease progression and mechanisms of infection in future comparative disease studies. This approach to biomarker discovery may help to inform biological processes in wildlife populations and provide alternatives to invasive or postmortem samples.

Methodology for Biological Sample Collection, Processing, and Storage in the Newcastle 1000 Pregnancy Cohort: Protocol for a Longitudinal, Prospective Population-Based Study in Australia.

Research in the developmental origins of health and disease provides compelling evidence that adverse events during the first 1000 days of life from conception can impact life course health. Despite many decades of research, we still lack a complete understanding of the mechanisms underlying some of these associations. The Newcastle 1000 Study (NEW1000) is a comprehensive, prospective population-based pregnancy cohort study based in Newcastle, New South Wales, Australia, that will recruit pregnant women and their partners at 11-14 weeks' gestation, with assessments at 20, 28, and 36 weeks; birth; 6 weeks; and 6 months, in order to provide detailed data about the first 1000 days of life to investigate the developmental origins of noncommunicable diseases. The study aims to provide a longitudinal multisystem approach to phenotyping, supported by robust clinical data and collection of biological samples in NEW1000. This manuscript describes in detail the large variety of samples collected in the study and the method of collection, storage, and utility of the samples in the biobank, with a particular focus on incorporation of the samples into emerging and novel large-scale "-omics" platforms, including the genome, microbiome, epigenome, transcriptome, fragmentome, metabolome, proteome, exposome, and cell-free DNA and RNA. Specifically, this manuscript details the methods used to collect, process, and store biological samples, including maternal, paternal, and fetal blood, microbiome (stool, skin, vaginal, oral), urine, saliva, hair, toenail, placenta, colostrum, and breastmilk. Recruitment for the study began in March 2021. As of July 2024, 1040 women and 684 partners were enrolled, with 922 infants born. The NEW1000 biobank contains 24,357 plasma aliquots from ethylenediaminetetraacetic acid (EDTA) tubes, 5284 buffy coat aliquots, 4000 plasma aliquots from lithium heparin tubes, 15,884 blood serum aliquots, 2977 PAX RNA tubes, 26,595 urine sample aliquots, 2280 fecal swabs, 17,687 microbiome swabs, 2356 saliva sample aliquots, 1195 breastmilk sample aliquots, 4007 placental tissue aliquots, 2680 hair samples, and 2193 nail samples. NEW1000 will generate a multigenerational, deeply phenotyped cohort with a comprehensive biobank of samples relevant to a large variety of analyses, including multiple -omics platforms. DERR1-10.2196/63562.

Factors Associated With Missing Biological Samples in the Dog Ageing Project.

The Dog Aging Project (DAP) is a longitudinal study examining aging in US companion dogs, where missing data pose a challenge to result validity and statistical power. Early recognition and procedural adjustments are vital to address missing samples. This research assesses missing biospecimen samples within the PRECISION cohort of the DAP, aiming to identify contributing factors and propose mitigation strategies. Ninety sample kits collected between August 2021 and October 2021 were evaluated for the missingness of individual kit components. A primary assessment reviewed the missingness of each sample type with hypothesized associated factors. A secondary assessment addressed the missingness of plasma and serum aliquots made after check-in. EDTA samples for flow cytometry, peripheral blood mononuclear cells (PBMCs), DNA and biobanking were missing at rates of 18.9%, 13.3%, 6.7% and 5.6%, respectively. Faecal samples were missing at 5.6%. Urine and plasma were each missing at 2.2%. Serum, hair and dried blood spot cards were each missing at 1.1%. No statistically significant difference in missingness was seen across regions, ease of blood collection, or cooperativeness of dog during sample collection. Higher time-to-receipt was associated with a higher missingness proportion. Increasing missingness proportions were seen across plasma and serum aliquots that were created in sequential order. The study suggests reevaluating sample collection instructions to address frequently missing sample types and prevent further loss. Investigation into collection procedures for these types is recommended to identify potential causes of sample yield loss.

Discrepancies of bovine haptoglobin concentrations between serum and plasma using two different anticoagulants and a colorimetric assay based on peroxidase activity.

Haptoglobin (Hp) is an emerging diagnostic marker in cattle, and knowledge of suitable sample types and measurement methods is important. The aims of this study were to compare the results of a colorimetric assay (CA) and an ELISA for bovine Hp using serum, EDTA plasma, and lithium-heparinized (LH) plasma, respectively, and to assess the diagnostic potential for puerperal metritis. In experiment 1, Hp was measured in pooled aliquots of serum (n = 10), EDTA plasma (n = 10), and LH plasma (n = 10) of 100 healthy fresh lactating dairy cows from 10 farms using both the CA and the ELISA. In experiment 2, five healthy and five cows with acute puerperal metritis were sampled, and Hp was determined using both assays for all three sample types. In experiment 3, aliquots of serum and LH plasma from cows in different lactation stages were transferred into plain, EDTA-coated, and LH-coated tubes and mixed before colorimetric analyses. Distilled water was also placed into each tube type and treated similarly. Plasma samples measured with the CA showed on average 2.3 (EDTA) and 2.5 (LH) times higher Hp concentrations compared with serum, whereas no differences were seen with the ELISA results between sample types. Based on a clinical cut-off value, both methods differentiated sick from healthy cows. Haptoglobin measurements with the ELISA were less precise compared with CA measurements due to high dilutions. No influence of the anticoagulants on the CA was observed. Due to measurement discrepancies between serum and plasma, CAs for bovine Hp based on peroxidase activity should be performed with serum, or specific reference ranges for plasma samples should be established. In this study, CA results obtained with LH plasma were more precise than results obtained with EDTA plasma. Both the CA and the ELISA are suitable diagnostic tools for the diagnosis of puerperal metritis, but CA measurements were more precise in this study.

Diagnostic serology test comparison for Q fever and Rift Valley fever in humans and livestock from pastoral communities.

Q fever (QF) and Rift Valley fever (RVF) are endemic zoonotic diseases in African countries, causing significant health and economic burdens. Accurate prevalence estimates, crucial for disease control, rely on robust diagnostic tests. While enzyme-linked immunosorbent assays (ELISA) are not the gold standard, they offer rapid, cost-effective, and practical alternatives. However, varying results from different tests and laboratories can complicate comparing epidemiological studies. This study aimed to assess the agreement of test results for QF and RVF in humans and livestock across different laboratory conditions and, for humans, different types of diagnostic tests. We measured inter-laboratory agreement using concordance, Cohen's kappa, and prevalence and bias-adjusted kappa (PABAK) on 91 human and 102 livestock samples collected from rural regions in Chad. The serum aliquots were tested using ELISA in Chad, and indirect immunofluorescence assay (IFA) (for human QF and RVF) and ELISA (for livestock QF and RVF) in Switzerland and Germany. Additionally, we examined demographic factors influencing test agreement, including district, setting (village vs. camp), sex, age, and livestock species of the sampled individuals. The inter-laboratory agreement ranged from fair to moderate. For humans, QF concordance was 62.5%, Cohen's kappa was 0.31, RVF concordance was 81.1%, and Cohen's kappa was 0.52. For livestock, QF concordance was 92.3%, Cohen's kappa was 0.59, RVF concordance was 94.0%, and Cohen's kappa was 0.59. Multivariable analysis revealed that QF test agreement is significantly higher in younger humans and people living in villages compared to camps and tends to be higher in livestock from Danamadji compared to Yao, and in small ruminants compared to cattle. Additionally, RVF agreement was found to be higher in younger humans. Our findings emphasize the need to consider sample conditions, test performance, and influencing factors when conducting and interpreting epidemiological seroprevalence studies.

B-040 Direct Aspiration of Cryogenic Matrix Tubes on the Siemens Atellica System With Epic-Beaker Integration of External Barcode IDs for Analysis Small Volume Biobanked Specimens

Abstract Background Most core chemistry and immunochemistry clinical analyzers are designed for direct sampling of primary blood tubes with volumes ranging from 3.5-10 mL. This limits analysis of low volume pediatric specimens and biobanked aliquoted samples from clinical trials without manual technologist intervention. This work outlines a microsampling and data transmission scheme for analysis of 500 uL aliquots of biobanked serum collected for the Nutrition for Precision Health study powered by the NIH All of Us Research Program without relabeling secondary aliquot tubes after receipt. Methods Commercial plastic clip bearings sourced from IGUS (LCM-08-02) were used to stabilize the matrix tube in a standard 5 mL Sarstedt aliquot tube. 96-well format Matrix Tubes (ThermoFisher 3719-11) with pre-printed 10-digit side barcodes were programmed in the Atellica software as a custom false bottom tube with specifications height 75 mm, diameter 12 mm, bottom offset 41 mm and were loaded using a dedicated false bottom tray. Manifest files (.csv) containing the sample ID, clinical trials participant ID, collection date/time, age and sex were ingested into Epic via an Incoming Orders HL7 interface. Non-human patient records were generated using the participant ID as an external patient ID and patient name and calculating the Date of Birth based on the age in years given in the manifest file. Test orders were created based on assigned testing for the sample ID in the manifest. The sample ID corresponding the preprinted Matrix Tube barcode ID was inserted into the Epic container identifier field. Results Test orders were automatically transmitted to the Atellica Data Manger (ADM) allowing specimens to be run on the Atellica System (CH and IM modules) and results transmitted to Epic utilizing the established Data Innovations (DI) interface and processes used for patient samples without the need for Epic generated labels. Successful scanning and recognition of Matrix Tube barcodes was 100%. Complete aspiration of a Comprehensive Metabolic Panel, Lipid Panel, Iron, TIBC, hsCRP, insulin, and H, I, L indices occurred for 50% of samples tested without intervention. Approximately 250 uL of the 500 uL aliquot remained after aspiration of all tests (247.7 uL total volume) from the Matrix Tube. No probe crashes were observed despite the narrower 8 mm diameter of the Matrix Tubes versus the standard 12- or 13-mm diameter of primary blood collection tubes. Conclusions This Epic-DI-ADM data workflow and microsampling adaptation for Matrix Tube aspiration allows greater container type flexibility for the receipt and analysis of external samples delivered in non-primary blood tubes for clinical laboratories. Future work involving adjustment of the false bottom tube offset depth may reduce unusable sample volume allowing for a higher percentage of complete aspiration without intervention. This work is significant because it suggests existing clinical analyzers are easily adaptable to microsampling smaller volume tubes without significant effort.

Collection of biospecimens from the inspiration4 mission establishes the standards for the space omics and medical atlas (SOMA)

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from four crew members longitudinally before (Launch: L-92, L-44, L-3 days), during (Flight Day: FD1, FD2, FD3), and after (Return: R + 1, R + 45, R + 82, R + 194 days) spaceflight, spanning a total of 289 days across 2021-2022. The collection process included venous whole blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies. Venous whole blood was further processed to obtain aliquots of serum, plasma, extracellular vesicles and particles, and peripheral blood mononuclear cells. In total, 2,911 sample aliquots were shipped to our central lab at Weill Cornell Medicine for downstream assays and biobanking. This paper provides an overview of the extensive biospecimen collection and highlights their processing procedures and long-term biobanking techniques, facilitating future molecular tests and evaluations.As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can aid future human spaceflight and space biology experiments.

Cohort profile of BIGPROMISE: a perioperative biobank of a high-risk surgical population

PurposePostoperative complications increase mortality, disability and costs. Advanced understanding of the risk factors for postoperative complications is needed to improve surgical outcomes. This paper discusses the rationale and profile of...

Assessing EPO stability in urine and comparing recombinant EPO detectability in matched urine, venous serum, and capillary serum following a controlled epoetin alfa administration.

The instability of erythropoietin receptor agonists (ERAs, i.e., EPO) in urine presents a challenge to their detectability in doping control samples; however, this issue is not often seen in blood (serum) samples. With the anti-doping field beginning to transition into alternative blood collection technologies, it is important to understand recombinant EPO (rEPO) detectability in serum samples collected from one such capillary collection device, the Tasso+ SST. Twelve individuals were administered a single, 40 IU/kg dose of rEPO (epoetin alfa, EPOGEN®). Following administration, matched urine, venous serum, and capillary serum samples were concurrently collected. Urine aliquots were subject to various storage times and temperatures mimicking shipping conditions of doping control urine samples to assess EPO stability, while other urine aliquots, venous serum, and capillary serum aliquots were frozen until analysis to understand rEPO detectability across all three matrices. EPO and rEPO instability was identified in urine collected from 8 of 12 participants, especially in aliquots stored at room temperature and 37°C. In some of these unstable samples, rEPO was still detectable, while in others, no recombinant nor endogenous EPO was detectable and would have resulted in negative sample reports. Analyzing the concurrently collected urine, venous, and capillary serum samples, rEPO detectability was identical across the three matrices. In most cases, rEPO was detectable for at least 168 h post-administration. Noting greater stability in blood compared with urine, it is recommended that anti-doping authorities utilize this novel capillary serum collection technology to improve overall ERA detectability in doping control samples.

EFFECT OF WHITE SUGAR, BROWN SUGAR AND JAGGERY POWDER SYRUP ON BIOCHEMICAL AND HISTOPATHOLOGICAL MARKERS OF SPRAGUE-DAWLEY RATS

Hypertension accounts for 10% of the total annual health budget in developed countries.1 By 2025, the number of people living with hypertension is expected to reach 1.56 billion people.2Dietary factors that increase blood pressure (BP) are of interest to public health authorities.3,4Twenty adult male Sprague-Dawley rats were taken. All groups received standard chow ad libitum. Controls (A), received plain drinking water, and the Sucrose treatment rats received 10% sucrose solution prepared daily for 12 weeks. Non-invasive blood pressure was recorded using computer based data recording system power lab model: M-ML 856, Australia with rat tail cuff attachment.Blood samples were collected. Serum aliquots were frozen at ?80°C for: ALT, AST, TG, total cholesterol (TC), and high-density lipoprotein (HDL) were determined using commercial enzymatic methods (CELM diagnosis, São Paulo, Brazil). The concentration of VLDL and low-density lipoprotein was measured. ANOVA and Post-Hoc Tukey tests were applied to determine statistical difference among the study groups. The results showed variable effects on biochemical and histopathological findings in rat liver, kidney and pancreas. The pancreas and kidneys of the rats showed adverse inflammatory histopathological changes maximally in rats taking jaggery syrup (Shakkar) flowed by white sugar and minimal in Shakkar. The adverse effects on liver cells were maximally seen on rats given brown sugar. This study enabled us to better understand the biochemical and histopathological effects of various types of sugars available in Pakistani market in order to create awareness regarding their effects on health.

Pre-analytical stability of selected biochemical analytes in serum of horses and oxen stored at -20°C.

Delays between blood collection and analysis are inevitable, and samples are always stored in the refrigerator. The current study aimed to evaluate the stability of serum total cholesterol (TC), triglycerides (TG), total protein (TP), albumin and urea (URA) in horses and oxen after storage at -20°C. Sera from apparently healthy 20 male horses and 20 oxen were obtained and aliquots of serum were divided into 3 portions. The first tube was used for baseline (T0) measurement of analyte values, whereas the other two tubes, T1 and T2, were stored at -20°C for 1 and 2 months, respectively, and analyte measurement was done. Results showed that the stability of TP (g/dL), URA (mg/dL) and TC (mg/dL) in oxen was statistically significant (p<0.05). In horses, the stability of URA (mg/dL), TP (g/dL) and TG (mg/dL) were also statistically significant (p<0.05). Additionally, URA and TC in oxen exceed TEa following measurement at T2 and TG in horses following measurement at T1 and T2. Laboratories should consider the storage temperature and time for specific analytes among animals. Therefore, stability studies at various storage temperatures and times are recommended to fully validate the stability of the analytes.

Biobanking in the hospital of a multidisciplinary research medical center as a potential for a wide research range. Part I. Organizational and methodological aspects

Aim. To develop an algorithm and manage total biobanking of samples of whole blood, serum and plasma of patients in the hospital of the National Medical Research Center for Therapy and Preventive Medicine in order to create a detailed biosample collection through the integration of electronic medical records and a biobank database.Material and methods. The study includes all patients admitted to the hospital of the National Medical Research Center for Therapy and Preventive Medicine in various departments who signed informed consent for biobanking. Clinical information is collected as part of patient examination and stored in electronic medical records in the Medialog medical information system. Biobanking of blood and related products is carried out in accordance with standard operating procedures. From each patient, 12 aliquots of serum and blood plasma are stored, as well as 1 tube of whole blood with ethylenediaminetetraacetic acid potassium salt. Information about biospecimens, patient identification numbers and storage location coordinates are contained in the biobank database.Results. An algorithm for total biobanking of biomaterial and data from inpatients has been developed and put into practice. This collection is associated with large biomedical data stored in a medical information system, laboratory information system and biobank. The combined databases make it possible to search for samples in the collection according to specified criteria.Conclusion. The collection developed can be used for a wide range of studies by forming patient samples according to the necessary criteria. The developed algorithm for total biobanking in a hospital can be used in various medical centers equipped with biobanks.

Serum metabolome differences associated with subclinical intramammary infection caused by Streptococcus agalactiae and Prototheca spp. in multiparous dairy cows

Mastitis is one of the most significant diseases in dairy cows and causes several economic losses. Somatic cell count (SCC) is often used as an indirect diagnostic tool for mastitis, especially for subclinical mastitis (SCM) where no symptoms or signs can be detected. Streptococcus agalactiae is one of the main causes of contagious mastitis, while Prototheca spp. is an alga inducing environmental mastitis that is not always correlated with increased milk SCC. The aim of this study was to evaluate the changes in the metabolomic profile of blood in relation to subclinical intramammary infection (sIMI) in dairy cows. In addition, differences due to the etiologic agent causing mastitis were also considered. Forty Holstein-Friesian dairy cows in mid and late lactation were enrolled in this study with a cross-sectional design. Based on the bacteriological examination of milk, the animals were divided into 3 groups: Group CTR (control group; n = 16); Group A (affected by SCM with IMI of Streptococcus agalactiae; n = 17); and Group P (affected by SCM with IMI of Prototheca spp.; n = 7). Blood samples were collected in tubes containing clot activator from jugular vein. The serum aliquot was stored until metabolomic analysis by 1H-NMR. Statistical analysis was conducted fitting a linear model with the group as fixed effect and SCC as covariate. Forty-two metabolites were identified and among them, 10 were significantly different among groups. Group A and P showed greater level of His and lactose, and lower level of acetate, Asn, and dimethylamine compared with Group CTR. Group A showed high level of Val, while the Group P showed also high level of Cit and methylguanidine, and lower level of 3-hydroxybutyrate, acetone, allantoin, carnitine, citrate, and ethanol. These metabolites were related to ruminal fermentations, energy metabolism, urea synthesis and metabolism, immune and inflammatory response, and mammary gland permeability. These results are suggestive of a systemic involvement of sIMI and that the metabolic profile of animals with SCM undergoes changes related to the etiological agent of mastitis.

Detection of early-stage cancers using circulating orphan non-coding RNAs in blood.

3051 Background: Orphan non-coding RNAs (oncRNAs) are a novel category of small RNAs (smRNAs) that are present in tumors and largely absent in healthy tissue. We investigated the utility of oncRNAs extracted from serum for early cancer detection across seven cancer types. Methods: We collected 2,882 serum samples from individuals with known cancers of the bladder ( n=152), breast (220), colon and rectum (141), kidney (283), lung (281), pancreas (287), and stomach (280) as well as donors with no history of cancer (1,238). We used 0.5 mL serum aliquots to generate and sequence smRNA libraries at an average depth of 20 million 50-bp single-end reads. Samples were split into age-, sex-, and smoking status-matched training (1,232 cancer; 922 control) and validation (412 cancer; 316 control) cohorts. A large catalog of oncRNAs specific to each cancer was created using tumor and adjacent normal samples from The Cancer Genome Atlas (TCGA) smRNA-seq database. Using TCGA-derived oncRNAs, we trained a machine learning model to predict cancer presence and tissue of origin (TOO) in a 5-fold cross validation setup using our training cohort. For the validation cohort, we averaged the predictions from the five training cohort models. Results: The model ROC-AUC for detecting cancer was 0.95 (95% CI: 0.94–0.95 for training and 0.94–0.97 for validation cohorts). Sensitivities for detecting cancer at 95% specificity were 0.74 (0.70–0.76) for early stage (I/II) and 0.80 (0.76–0.84) for late stage (III/IV) cancers in the training cohort, and 0.77 (0.71–0.81) and 0.81 (0.73–0.87) in the validation cohort. Sensitivities of detection for each cancer type are shown. For samples with cancer and TOO predictions, our top 1 and top 2 TOO accuracy was 0.76 (0.68–0.84) and 0.83 (0.76–0.90) for the validation set. Conclusions: These results demonstrate that oncRNAs detected in serum can be used for accurate, early detection, and localization of multiple cancers. [Table: see text]

Vitamin D deficiency in pregnancy and the risk of preterm birth: a nested case–control study

BackgroundEach year, an estimated 15 million babies are born preterm. Micronutrient deficiencies, including vitamin D deficiency (VDD), are common in many low- and middle-income countries (LMICs), and these conditions are often associated with adverse pregnancy outcomes. Bangladesh experiences a high prevalence of VDD. The country also has a high preterm birth (PTB) rate. Using data from a population-based pregnancy cohort, we estimated the burden of VDD during pregnancy and its association with PTB.MethodsPregnant women (N = 3,000) were enrolled after ultrasound confirmation of gestational age at 8–19 weeks of gestation. Trained health workers prospectively collected phenotypic and epidemiological data at scheduled home visits. Trained phlebotomists collected maternal blood samples at enrollment and 24 -28 weeks of gestation. Aliquots of serum were stored at -800 C. We conducted a nested case–control study with all PTB (n = 262) and a random sample of term births (n = 668). The outcome, PTB, was defined as live births < 37 weeks of gestation, based on ultrasound. The main exposure was vitamin D concentrations of 24–28 weeks maternal blood samples. The analysis was adjusted for other PTB risk factors. Women were categorized as VDD (lowest quartile of 25(OH)D; < = 30.25 nmol/L) or not deficient (upper-three quartiles of 25(OH)D; > 30.25 nmol/L). We used logistic regression to determine the association of VDD with PTB, adjusting for potential confounders.ResultsThe median and interquartile range of serum 25(OH)D was 38.0 nmol/L; 30.18 to 48.52 (nmol/L). After adjusting for co-variates, VDD was significantly associated with PTB [adjusted odds ratio (aOR) = 1.53, 95% confidence interval (CI) = 1.10 – 2.12]. The risk of PTB was also higher among women who were shorter (aOR = 1.81, 95% CI: 1.27–2.57), primiparous (aOR = 1.55, 95% CI = 1.12 – 2.12), passive smokers (aOR = 1.60, 95% CI = 1.09 – 2.34), and those who received iron supplementation during pregnancy (aOR = 1.66, 95% CI: 1.17, 2.37).ConclusionVDD is common in Bangladeshi pregnant women and is associated with an increased risk of PTB.

Blue Tongue Virus in Dairy Cattle of Minas Gerais, Brazil - Serological Survey

Background: Blue tongue (BT) is a noncontagious viral disease transmitted by hematophagous arthropods, especially of the genus Culicoides. The economic impact of the disease is related not only to deaths in sheep herds but also to the possible correlation of virus infection with the development of other diseases, such as pneumonia, abortion and movement problems. The economic losses caused by Blue Tongue are linked to restrictions on the import and export of animals and their genetic material and to the reproductive disorders associated with this disease. In addition, the fact that cattle take the role of reservoir, combined with the care by other countries with outbreaks of infection and biological contamination of their products, hinders trade in Mercosul, United States and Europe. Cattle are affected by Blue Tongue Virus in endemic areas and in some epidemic areas, but the development of clinical disease is rare. The clinical signs, when evident, range from reproductive losses, such as embryonic death, abortion, fetal malformation, temporary sterility, infertility in bulls, stillbirths and the birth of weak animals. The objective of this study was to determine the epidemiological aspects of Blue Tongue Virus (BTV) infection in dairy cattle in the Lavras region, state of Minas Gerais, Brazil. Materials, Methods &amp; Results: A cross-sectional study was conducted to evaluate the frequency of cattle and herds seropositive for Blue Tongue in the southern region of Minas Gerais. In this study, 54 dairy farms were visited. A total of 586 serum samples were collected from female cattle of reproductive age. Sampling was random, and serum samples were collected from lactating cows over 24 months of age by puncture of the jugular vein and/or coccidian vein. The samples were transported and stored at the Setor de Patologia Veterinária, at the Universidade Federal de Lavras (SPV-UFLA), where they were centrifuged, and the serum aliquots were obtained, transferred to microtubes and kept at -20 °C until the serological tests were performed. The samples were tested with the agarose gel immunodiffusion test (AGID) for anti-blue tongue virus antibodies. The AGID test is more practical and is the main method used to identify Blue Tongue Virus seroprevalence in different ruminant species. They are considered important tools for epidemiological surveillance of the disease. A prevalence of 83.28% was observed among animals that were seropositive for Blue Tongue Virus (488/586; IC 95% = 80.0 - 86.21). In addition, 100% (54/54; IC 95% = 93.4 - 100.0) of the farms had at least one positive animal, with rates ranging from 45.45% to 100% within the herds and where 22.22% of the farms had rates of 100% of the animals being positive. Discussion: Blue Tongue is a disease known to affect domestic and wild ruminants in Brazil. However, there is a lack of more precise information about its epidemiology and occurrence in the country and of joint efforts of researchers, producers and the government to understand in detail both the biology of vectors and the viral biology of Blue Tongue Virus in Brazil. This is the first record of detection of anti-blue tongue virus antibodies in cattle in the southern region of Minas Gerais. The results suggest that Blue Tongue Virus is present in cattle in the study area. Keywords: BTV, prevalence, AGID, anti-BTV antibodies, Orbivirus, Reoviridae.

Stability of Some Biochemical Parameters in Sheep and Goat Serum Stored at -20℃.

In Veterinary Medicine biochemicalinvestigation of serumis widely used to aid diagnosis and treatment. However, delays usually happen between sampling and analysis. As a result, the serum is stored in refrigerators.In this regard, information on the effects of temperature and storage duration on the stability of the analyte is incomplete in general and its effect in sheep and goat serum is not described. Therefore, the objective of this study was to examine the stability of selected biochemical analytes from sheep and goat serum following storage at -20℃ for 2 months. Serum from 20 apparently healthy male 2-2.5 year-old sheep and goats was obtained and aliquots of serum from each sample were kept in three tubes. The first tube is for baseline (T0), which is done within an hour, while the other two (T1 and T2) are stored at -20℃ for 1 and 2 months, respectively. Total protein, albumin, urea, total cholesterol, and triglycerides were assayed. The results revealed that storage temperature and duration for up to 2 months had no significant effect on any analytes except for urea in goats. The changes in terms of total observed error (TEo) for total protein; albumin and urea were greater than the acceptable values in both animals. Thus, further studies are required to assure alteration of analyte at various storage temperatures and duration. In addition, implementation of quality systems to achieve quality targets for analytes with greater TEo as compared to the established TEa is needed.

A comprehensive UHPLC-MS/MS method for the analysis of endogenous and exogenous steroids in serum for anti-doping purposes.

In the context of steroid analyses, the use of blood could represent a valuable complement to urine. While the blood steroid profile is currently being established to aid unveiling testosterone (T) doping, this matrix is also well suited for detection of exogenous anabolic steroids and steroid esters. In this study, a method to determine a simplified blood steroid profile in combination with the direct detection of exogenous anabolic steroids and steroid esters using just one serum aliquot was developed to obtain a comprehensive analytical workflow. Following the first chromatographic analysis of endogenous and exogenous steroids, samples were derivatised with Girard's reagent T (GT) to improve the ionisation of steroid esters and re-injected. The quantitative performance for T, androstenedione (A4) and 5α-dihydrotestosterone (DHT) was evaluated and the method was validated for qualitative analysis of exogenous analogues with estimated limits of detection (LOD) between 50 and 500 pg/ml. To demonstrate the applicability of the method, samples collected from a clinical study with an oral administration of testosterone undecanoate (TU) to 19 male volunteers were then analysed. The individual serum steroid profiles with the endogenous markers T, A4 and DHT were established as well as the concentrations of TU. TU was detected in all 19 volunteers up to 24 h, while DHT represented the most promising biomarker in endogenous steroid profile for the detection of oral TU administration. These results showed that the selected approach to combine exogenous and endogenous steroid analysis has the potential to strengthen T doping detection in the future.

Abstract 1476: Stability of circulating microRNA for plasma and serum biomarker studies

Abstract Purpose: To verify the stability and reliability of circulating microRNA (miRNA) profiles in plasma and serum under different processing and storage conditions for the optimization of future biomarker analyses. Background: Development of blood-based early cancer detection methods have become increasingly popular in recent years. RNA profiles have been investigated; however, obtaining reliable RNA data has been a challenge due to rapid degradation by RNases. miRNAs are short non-coding regulatory RNAs that are more stable as they are complexed with proteins or packaged in exosomes. Thus, circulating miRNA profiles have the potential to serve as a diagnostic test for cancer. Optimization of collection protocols is a crucial next step for widespread use. Methods: Whole blood was drawn after Institutional Review Board approval. Blood was processed into either plasma or serum aliquots. The samples were stored at different temperatures (0 or 25°C) for varying periods of time (0-24 hours). miRNA was extracted using a Qiagen miRNeasy Serum/Plasma kit. Changes in profiles were assessed with RT-qPCR using a previously established panel of consistently expressed miRNAs (miR15b, miR16, miR21, miR24, miR223). Values were compared using two-tailed t-tests and values are presented as mean ± standard deviation. Results: Mean Cq values were similar at 0 and 24 hours when serum was stored on ice, suggesting negligible miRNA degradation: miR15b (29.6±0.5 vs 29.8±0.4, p=0.11), miR16 (23.5±0.8 vs 23.3±1.0, p=0.55), miR21 (28.6±0.5 vs 28.9±0.6, p=0.17), miR24 (27.4±0.7 vs 27.0±0.5, p=0.09), and miR223 (22.9±0.5 vs 22.9±0.7, p=0.90). There was similar stability over time for 60% of tested miRNAs when serum was left at room temperature: miR15b (30.3±0.7 vs 30.7±0.9, p=0.22), miR16 (23.9±0.7 vs 24.3±1.0, p=0.28), and miR21 (30.0±0.6 vs 29.9±0.6, p=0.38). Two of the miRNAs demonstrated a statistically significant increase in mean Cq after 24 hours: miR24 (27.6±0.7 vs 28.2±0.7, p=0.03) and miR223 (23.4±0.4 vs 24.6±0.6, p=&amp;lt;0.01). In general, these trends were similar when plasma was collected, as compared to serum, at both room temperature and on ice. miRNA profiles collected from serum were overall like those obtained from plasma. Conclusions: These data confirm and expand upon past reports demonstrating the remarkable stability of miRNA. Even in suboptimal processing conditions (i.e., room temperature for 24 hours), the miRNA profile is remarkably consistent. Although blood processing and transport times may vary between institutions, our data suggest these discrepancies may not affect the efficacy of miRNA as a blood biomarker. We plan to validate our findings using bulk small RNA sequencing, which we hypothesize will similarly demonstrate minimal changes in the entire miRNA landscape in different conditions. We will then utilize our institutional biorepository to identify potentially unique miRNA profiles present in patients with cancer. Citation Format: Erryk S. Katayama, Jonathan J. Hue, Mehrdad Zarei, Hallie J. Graor, Omid Hajihassani, Ali Vaziri-Gohar, Jordan M. Winter. Stability of circulating microRNA for plasma and serum biomarker studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1476.