Reference Blood Samples Research Articles

A novel technique for estimating age and demography of long-lived seabirds (genus Pterodroma) using an epigenetic clock for Gould's petrel (Pterodroma leucoptera).

Understanding the demography of wildlife populations is a key component for ecological research, and where necessary, supporting the conservation and management of long-lived animals. However, many animals lack phenological changes with which to determine individual age; therefore, gathering this fundamental information presents difficulties. More so for species that are rare, highly mobile, migratory and those that reside in inaccessible habitats. Until recently, the primary method to measure demography is through labour intensive mark-recapture approaches, necessitating decades of effort for long-lived species. Gadfly petrels (genus: Pterodroma) are one such taxa that are overrepresented with threatened and declining species, and for which numerous aspects of their ecology present challenges for research, monitoring and recovery efforts. To overcome some of these challenges, we developed the first DNA methylation (DNAm) demography technique to estimate the age of petrels, using the epigenetic clock of Gould's petrels (Pterodroma leucoptera). We collected reference blood samples from known-aged Gould's petrels at a long-term monitored population on Cabbage Tree Island, Australia. Epigenetic ages were successfully estimated for 121 individuals ranging in age from zero (fledgling) to 30 years of age, showing a mean error of 2.24 ± 0.17 years between the estimated and real age across the population. This is the first development of an epigenetic clock using multiplex PCR sequencing in a bird. This method enables demography to be measured with relative accuracy in a single sampling trip. This technique can provide information for emerging demographic risks that can mask declines in long-lived seabird populations and be applied to other Pterodroma populations.

Exploration of identifying individual tumor tissue based on probabilistic model.

Variations in the tumor genome can result in allelic changes compared to the reference profile of its homogenous body source on genetic markers. This brings a challenge to source identification of tumor samples, such as clinically collected pathological paraffin-embedded tissue and sections. In this study, a probabilistic model was developed for calculating likelihood ratio (LR) to tackle this issue, which utilizes short tandem repeat (STR) genotyping data. The core of the model is to consider tumor tissue as a mixture of normal and tumor cells and introduce the incidence of STR variants (φ) and the percentage of normal cells (Mxn) as a priori parameters when performing calculations. The relationship between LR values and φ or Mxn was also investigated. Analysis of tumor samples and reference blood samples from 17 colorectal cancer patients showed that all samples had Log 10(LR) values greater than 1014. In the non-contributor test, 99.9% of the quartiles had Log 10(LR) values less than 0. When the defense's hypothesis took into account the possibility that the tumor samples came from the patient's relatives, LR greater than 0 was still obtained. Furthermore, this study revealed that LR values increased with decreasing φ and increasing Mxn. Finally, LR interval value was provided for each tumor sample by considering the confidence interval of Mxn. The probabilistic model proposed in this paper could deal with the possibility of tumor allele variability and offers an evaluation of the strength of evidence for determining tumor origin in clinical practice and forensic identification.

New polymer brush-coated monodisperse magnetic nanoparticles prepared via interface-mediated RAFT polymerization for high-throughput DNA extraction from pathogen bacteria

Polymerase chain reaction (PCR) is gold-standard method for molecular detection of pathogens. DNA extraction from a complex biological medium is a crucial process for PCR based detection. Recently, magnetic nanoparticles-based methods are of great interest for genetic material isolation. In our work, we offer new magnetic nanoparticles based high-throughput DNA extraction method for pathogen detection in biofluids. For this purpose, first time polymer brush coated monodisperse magnetic nanoparticles were prepared with interface-mediated RAFT polymerization to be used in DNA extraction process. After synthesizing Fe3O4 nanoparticles, silica shell was coated on the magnetic nanoparticle core. Polymer brushes was immobilized on the amine functionalized silica layer using RAFT agent. Subsequently, the resulting particles with poly(vinylbenzyltrimethylammonium chloride) (PVBTAC) grafted were finalized through interface-mediated RAFT polymerization. The functional groups on the nanoparticles were characterized using FTIR spectroscopy. Size distribution and zeta potential analysis of the particles were performed by TEM and Zeta-sizer respectively. Adsorption and recovery studies of plasmid DNA were carried out on the polymer-coated magnetic nanoparticles. The developed particle provided both the rapid extraction of DNA and high-yield DNA recovery without requiring extended incubation and elution time. The maximum adsorption capacity was determined as 238.1 μg/mg. About 90 % of the input DNA was recovered. The total time of DNA extraction took just 20 min. We have successfully demonstrated the extraction of dsDNA of Enterococcus faecalis spiked to reference blood sample using the developed magnetic particles. The extracted plasmid DNA and dsDNA were confirmed by gel electrophoresis and qPCR methods respectively. The results showed that the proposed magnetic iron oxide nanoparticles (MIONp) will be a strong candidate for DNA extraction process from biological matrices.

Detection of doping control sample substitutions via single nucleotide polymorphism-based ID typing.

The authenticity of a doping control sample is a key element of sports drug testing programmes. Doping control sample manipulation by providing another individual's urine or blood (instead of the tested athlete's sample) has been observed in the past and is an unequivocal violation of the World Anti-Doping Agency anti-doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)-based sample authentication with a multi-target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, 3 gender markers and 5 quality control markers for DNA-profile determination. Sample analysis is based on a multiplex polymerase chain reaction step followed by a multiplex single base extension (SBE) reaction and subsequent SBE-product detection by MALDI-TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8ml) and DBS (20 μl) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 ml. Samples can be processed in a high-throughput format with an overall assay turnaround time of approximately 11 h. SNP-based DNA typing via MALDI-TOF MS thus represents a high throughput-capable possibility for doping control sample authentication. SNP profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings.

Internal validation of DNA extraction from human blood reference samples using Chelex 100 resin

O processo de extração de DNA é uma etapa crucial para o exame de identificação genética. O presente estudo tem por objetivo verificar a eficiência do protocolo de extração por meio da resina Chelex® 100 em amostras de referência de sangue com vistas à obtenção de DNA em quantidade e qualidade suficientes para a genotipagem, sem a necessidade de quantificação prévia. Foram utilizadas amostras de sangue total de 6 (seis) voluntários e, posteriormente, coletados suabes (por meio de imersão em tubo com sangue) para proceder à metodologia de extração por Chelex® 100. Em seguida, os produtos de extração foram quantificados, efetuou-se o cálculo da média e desvio padrão dos valores encontrados e, logo após, realizou-se a amplificação de todos os eluídos com o volume médio obtido no cálculo. Foi analisada a qualidade dos eletroferogramas das amostras amplificadas e calculada a taxa de retrabalho para a amostragem do estudo (razão do número de amostras com necessidade de reamplificação pelo número de amostras totais). Os resultados encontrados demonstraram que a utilização de 1 µL do DNA extraído por Chelex® 100, sem diluição, foi suficiente para a obtenção de perfis genéticos interpretáveis. Por fim, foi possível demonstrar que a etapa experimental da validação da extração de DNA a partir de suabes com sangue de referência por Chelex® 100 encontra-se concluída no Serviço de Perícias em Genética Forense da Polícia Federal, podendo, assim que completada as demais etapas documentais da validação, ser realizada com celeridade e eficiência, uma vez que o DNA obtido apresentou quantidade e qualidade suficientes para ser amplificado e genotipado, de acordo com os requisitos do Laboratório.

Agreement of somatic and renal near-infrared spectroscopy with reference blood samples during a controlled hypoxia sequence: a healthy volunteer study

Purpose: O3® Regional Oximetry (Masimo Corporation, California, USA) is validated for cerebral oximetry. We aimed to assess agreement of somatic and renal near-infrared spectroscopy with reference blood samples. Methods: O3 sensors were placed bilaterally on the quadriceps and flank of 26 healthy volunteers. A stepped, controlled hypoxia sequence was performed by adding a mixture of nitrogen and room air to the breathing circuit. O3-derived oxygen saturation values were obtained at baseline and at six decremental saturation levels (5% steps). Blood samples (radial artery, iliac vein (somatic reference) and renal vein) were obtained at each step. Reference values were calculated as: 0.7 × venous saturation + 0.3 × arterial saturation. The agreement between O3-derived values with blood reference values was assessed by calculating root-mean-square error accuracy and Bland-Altman plots. Results: The root-mean-square error accuracy was 6.0% between quadriceps oxygen saturation and somatic reference values. The mean bias was 0.8%, with limits of agreement from -7.7 to 9.3%. These were 5.1% and 0.6% (-8.3 to 9.5%) for flank oxygen saturation and somatic reference values, respectively, and 7.7% and -4.9% (-15.0 to 5.2%) for flank oxygen saturation and renal reference values. The kidney depth was 3.1 ± 0.9 cm below the skin. Conclusion: O3 regional oximetry can be used on the quadriceps and flank to monitor somatic saturation, yet has a saturation-level dependent bias. O3-derived values obtained at the flank underestimated renal reference values. Additionally, it is unlikely that the flank sensors did directly measure renal tissue. Trial registration: Clinicaltrials.gov (NCT04584788): registered October 6th, 2020.

A high-throughput platform for the rapid screening of vitamin D status by direct infusion-MS/MS

Vitamin D is an important fat-soluble prohormone with pleiotropic effects on human health, such as immunomodulation of the innate and adaptive immune system. There is an unmet clinical need for a rapid screening platform for 25-hydroxyvitamin D (25OH-D) determination without chromatographic separation that offers better precision and accuracy than immunoassays. Here, we introduce a high-throughput method for assessing vitamin D status from blood specimens based on direct infusion-MS/MS (DI-MS/MS) following click derivatization using 2-nitrosopyridine. We developed an optimized liquid-phase extraction protocol to minimize ion suppression when directly infusing serum or plasma extracts via a capillary electrophoresis system for quantitative determination of 25OH-D. Acceptable reproducibility (mean coefficient of variation = 10.9%, n = 412), recovery (mean = 102% at 15, 30, and 45 nmol/l), and linearity (R2 > 0.998) were achieved for 25OH-D with lower detection limits (limit of detection ∼1.2 nmol/l, S/N ∼ 3), greater throughput (∼3 min/sample), and less bias than a commercial chemiluminescence immunoassay prone to batch effects. There was mutual agreement in 25OH-D concentrations from reference blood samples measured by DI-MS/MS as compared with LC-MS/MS (mean bias = 7.8%, n = 18). We also demonstrate that this method could reduce immunoassay misclassification of vitamin D deficiency in a cohort of critically ill children (n = 30). In conclusion, DI-MS/MS offers a viable alternative to LC-MS/MS for assessment of vitamin D status in support of large-scale studies in nutritional epidemiology as well as clinical trials to rapidly screen individual patients who may benefit from vitamin D supplementation.

A single direct amplification method for forensic casework references on a variety of substrates

A single direct amplification method for buccal and blood reference samples, on different substrates, containing a wide range of cellular material, was developed. This method uses the GlobalFiler™ STR kit, which was designed for amplification of previously purified and quantified DNA. Buccal samples on cotton swabs, Bode collectors, and Whatman® FTA® cards, and liquid blood, blood on Whatman® 903 and FTA® cards, and autopsy bloods were examined. A first pass success rate, that is, complete concordant profiles with no GeneMapper™ ID-X quality flags, of > 95% was obtained. This method involves the lysis of reference samples (tips of cotton swabs, double 4.8 mm diameter Bode or FTA® buccal punches, and single 4.8 mm diameter blood punches on Whatman® 903 or FTA® cards) in 100 µl of Prep-n-Go™ lysis buffer, and amplification of 2 µl of the lysate with 25 cycles. Data collection is optimized by use of the spectral pull-up reduction and off-scale recovery features of the AB3500 Genetic Analyzer Data Collection version 4 software, and by injection of PCR products for two different lengths of time (15 and 7 s). Data was analyzed with GeneMapper™ ID-X version 1.6, which allowed an increased signal dynamic range (maximum peak height of 64000 RFU), and by employing a 10% global filter, and a 20% global stutter filter. Complete GlobalFiler™ profiles were generated from as little as 5 µl of blood, 1 buccal swipe on cotton swab and Bode collectors, and collection of buccal cells deposited on the foam heads of EasiCollect™ FTA® collectors for 5 s.

Epigenome-Wide Association Study of Acute Lymphoblastic Leukemia in Children with Down Syndrome

Background: Down syndrome (DS) is associated with an up to 30-fold increased risk of B-cell acute lymphoblastic leukemia (ALL), and DS-ALL patients have worse overall survival and increased long-term treatment-related health conditions compared with non-DS ALL patients. In a recent genome-wide association study of DS-ALL, established ALL genetic risk loci were associated with DS-ALL, with several single nucleotide polymorphisms (SNPs) conferring a larger effect on ALL risk in the context of DS than in euploidy. We performed an epigenome-wide association study (EWAS) to elucidate whether epigenetic differences at birth are associated with risk of subsequent DS-ALL.Methods: The DS-ALL Discovery Study included 147 DS-ALL cases and 198 DS controls from the International Study of Down Syndrome Acute Leukemia, with newborn dried bloodspots (DBS) obtained from California (n=326) and Washington state (n=19) biobanks. The DS-ALL Replication Study included 24 DS-ALL cases and 24 DS controls with newborn DBS from the Michigan Neonatal Biobank. DNA was isolated from DBS, bisulfite converted, and assayed using Illumina Infinium MethylationEPIC Beadchip genome-wide DNA methylation arrays. Raw data were processed using “minfi” and “noob” packages in R. Reference-based deconvolution of blood cell proportions was performed using the Identifying Optimal DNA methylation Libraries (IDOL) algorithm, using DNA methylation data from cord blood reference samples, to estimate proportions of B cells, T cells (CD4+ and CD8+), monocytes, granulocytes, natural killer cells, and nucleated red blood cells. We compared each cell type proportion between DS-ALL cases and DS controls using linear regression adjusting for sex, plate, and principal components (PCs) to account for genetic ancestry. To identify single CpG probes associated with DS-ALL risk, we performed a multiethnic EWAS of DS-ALL in each study using linear regression adjusting for sex, plate, and PCs related to: 1) cell-type proportions and 2) genetic ancestry. Differentially methylated regions (DMRs) were identified using DMRcate and comb-p methods. In the Discovery Study, genome-wide SNP array data were available for 131 cases and 130 controls, and data from targeted sequencing of somatic mutations in exons 2/3 of GATA1 were available for 184/198 DS controls.Results: Deconvolution of blood cell proportions in the DS-ALL Discovery Study showed significantly higher B cell proportions in newborns with DS who later developed ALL (mean=0.0128, sd=0.0151) compared with DS controls (mean=0.00826, sd=0.0115) (P=6.4x10 -4, coefficient=0.0052). A significantly higher B cell proportion at birth was also found in DS-ALL cases in the independent Replication Study (cases mean=0.048, sd=0.024; controls mean=0.039, sd=0.028; P=0.03, coefficient=0.015). In the Discovery Study, the B cell difference remained significant (P=5.8x10 -3) with a similar effect size (coefficient=0.0045) after removal of GATA1 mutation-positive DS controls (n=30). We also investigated whether DS-ALL risk SNPs at ARID5B, IKZF1, GATA3, and CDKN2A may confound the association, but the increased B cell proportions in DS-ALL remained significant and effect estimates slightly increased in SNP genotype-adjusted models (coefficient range:0.0055-0.0059). In the EWAS of DS-ALL, 9 CpGs reached epigenome-wide significance (P<7.67x10 -8), including 2 CpGs overlapping the promoter of the tumor suppressor gene TRIM13, frequently deleted in B-CLL, although none of these showed evidence of association (P<0.05) in the Replication Study. We identified 125 DMRs associated with DS-ALL in the Discovery Study. For 3 DMRs, overlapping genes HOPX, SMIM24, and PPP1R10, all implicated in normal and leukemic stem cell function, there were multiple significant CpGs in the Replication Study (P<0.05) all with effects in the same direction as the Discovery Study DMRs.Conclusions: Increased B cell proportions in newborns with DS may be a risk factor for development of DS-ALL in childhood. This finding, based on DNA methylation data, requires confirmation using conventional cell count measures, and should be explored as a novel biomarker for ALL risk in the non-DS population. Single CpGs and DMRs associated with DS-ALL risk in our Discovery Study require further investigation, including in additional ALL case-control studies in DS and non-DS populations. DisclosuresMa: Celgene/Bristol Myers Squibb: Consultancy, Research Funding.

Lateral flow assays for the detection of African swine fever virus antigen are not fit for field diagnosis of wild boar carcasses.

African swine fever (ASF) is one of the most important viral diseases of domestic pigs and wild boar. Apart from endemic cycles in Africa, ASF is now continuously spreading in Europe and Asia. As ASF leads to severe but unspecific clinical signs and high lethality, early pathogen detection is of utmost importance. Recently, 'point-of-care' (POC) tests, especially immunochromatographic assays, have been intensively discussed for the use in remote areas but also in the context of on-farm epidemiological investigations and wild boar carcass screening. The later topic was the starting point for our present study. In detail, we evaluated the performance of the commercially available INGEZIM ASFV CROM Ag lateral flow assay (Eurofins Technologies Ingenasa) with selected high-quality reference blood samples, and with blood samples from wild boar carcasses collected under field conditions in Germany. While we observed a sensitivity of roughly 77% in freeze-thawed matrices of close to ideal quality, our approach to simulate field conditions in direct testing of blood samples from carcasses without any modification, resulted in a drastically reduced sensitivity of only 12.5% with the given sample set. Freeze thawing increased the sensitivity to roughly 44% which mirrored the overall sensitivity of 49% in the total data set of wild boar carcass samples. A diagnostic specificity of 100% was observed. In summary, the antigen LFA should not be regarded as a substitute for any OIE listed diagnostic method and has very limited use for carcass testing at the point of care. For optimized LFA antigen tests, the sensitivity with field samples must be significantly increased. An improved sensitivity is seen with freeze-thawed samples, which may indicate problems in the accessibility of ASFV antigen that could be overcome, to a certain extent, with assay modifications.

Allogeneic Hematopoietic Stem Cell Transplantation in Fanconi Anemia

Objective: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the treatment of choice in patients with Fanconi Anemia (FA). The aim of our study is to evaluate the impact and benefits of allogenic matched donor HSCT in a case of a 12 year-old girl with FA, who displayed good clinical evolution following 2 months post-transplantation. Patients and Methods: In the pre-transplant phase, reference blood samples from the donor and recipient were collected on EDTA. The DNA from blood samples was extracted using an automated Maxwell® 48 RSC instrument (Promega, USA) with the Maxwell® RSC Whole blood DNA kit (Promega, USA). For DNA quantification, the PowerQuant System kit (Promega, USA) was used with the ABI 7500 Real-time PCR system (Applied Biosystems, USA). The amplification of the short tandem repeat markers was performed using the 24plex Investigator QS kit (Qiagen, Germany) on a ProFlex PCR System. Furthermore, the PCR products were separated and detected on an ABI 3500 Genetic Analyzer (Applied Biosytems, USA). Results: After 30 days of the transplantation, a Complete Chimerism (CC) was achieved with a full replacement by donor derived hematopoietic cells. After 60 days of the transplantation, the CC status was maintained with improvement of hematological findings. Conclusion: In FA, chimerism monitoring after HSCT provides useful informations of engraftment or possibility of post-transplantation complications such as graft versus host disease.

Ultrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers.

Immune repertoire sequencing of the T-cell receptor can identify clonotypes that have expanded as a result of antigen recognition or hematological malignancies. However, current sequencing protocols display limitations with nonuniform amplification and polymerase-induced errors during sequencing. Here, we developed a sequencing method that overcame these issues and applied it to γδ T cells, a cell type that plays a unique role in immunity, autoimmunity, homeostasis of intestine, skin, adipose tissue, and cancer biology. The ultrasensitive immune repertoire sequencing method used PCR-introduced unique molecular identifiers. We constructed a 32-panel assay that captured the full diversity of the recombined T-cell receptor delta loci in γδ T cells. The protocol was validated on synthetic reference molecules and blood samples of healthy individuals. The 32-panel assay displayed wide dynamic range, high reproducibility, and analytical sensitivity with single-nucleotide resolution. The method corrected for sequencing-depended quantification bias and polymerase-induced errors and could be applied to both enriched and nonenriched cells. Healthy donors displayed oligoclonal expansion of γδ T cells and similar frequencies of clonotypes were detected in both enrichment and nonenriched samples. Ultrasensitive immune repertoire sequencing strategy enables quantification of individual and specific clonotypes in a background that can be applied to clinical as well as basic application areas. Our approach is simple, flexible, and can easily be implemented in any molecular laboratory.

CCNB2 and AURKA overexpression may cause atypical mitosis in Japanese cortisol-producing adrenocortical carcinoma with TP53 somatic variant.

Many genomic analyses of cortisol-producing adrenocortical carcinoma (ACC) have been reported, but very few have come from East Asia. The first objective of this study is to verify the genetic difference with the previous reports by analyzing targeted deep sequencing of 7 Japanese ACC cases using next-generation sequencing (NGS). The second objective is to compare the somatic variant findings identified by NGS analysis with clinical and pathological findings, aiming to acquire new knowledge about the factors that contribute to the poor prognosis of ACC and to find new targets for the treatment of ACC. DNA was extracted from ACC tissue of seven patients and two reference blood samples. Targeted deep sequencing was performed using the MiSeq system for 12 genes, and the obtained results were analyzed using MuTect2. The hypothesis was obtained by integrating the somatic variant findings with clinical and pathological data, and it was further verified using The Cancer Genome Atlas (TCGA) dataset for ACC. Six possible pathogenic and one uncertain significance somatic variants including a novel PRKAR1A (NM_002734.4):c.545C>A (p.T182K) variant were found in five of seven cases. By integrating these data with pathological findings, we hypothesized that cases with TP53 variants were more likely to show atypical mitotic figures. Using TCGA dataset, we found that atypical mitotic figures were associated with TP53 somatic variant, and mRNA expression of CCNB2 and AURKA was significantly high in TP53 mutated cases and atypical mitotic figure cases. We believe this is the first report that discusses the relationship between atypical mitotic figures and TP53 somatic variant in ACC. We presumed that overexpression of CCNB2 and AURKA mRNA may cause atypical mitosis in TP53 somatic mutated cases. Because AURKA is highly expressed in atypical mitotic cases, it may be an appropriate indicator for AURKA inhibitors.

Salivary DNA Analysis: A Proof of Evidence

A number of evidences are found on the scene of crime, of which some prove to be the sole proof. In the present case, a steel cup found at the scene of murder proved to be the only evidence that could link the murder to the scene of crime. The steel cups were swabbed using the ‘Double-swab technique’ and profiles were generated. DNA profiles obtained from reference blood samples of suspect matched with DNA on the evidence found at the scene of crime.

Effect of the number of incisions and use of local anesthesia on the physiological indicators of surgically-castrated piglets

The objective of this study was to determine the effect of local anesthesia and the number of incisions performed on the physiological blood profile of piglets after surgical castration. A total of 60 male piglets were divided into five groups of 12 each, based on the surgical method employed and the use, or not, of local anesthesia, as follows: surgical castration using one horizontal incision in both testicles with (C1+L) and without (C1) local anesthesia; surgical castration using two vertical scrotal incisions with and without local anesthesia (C2+L and C2); and control piglets which were removed from their pens and held head-down by their hind limbs for approximately 90 s to simulate castration (SIM). Reference blood samples were drawn 24 h before castration (RV), immediately after surgery or simulated castration (PC), and at 24 and 48 h post-castration, to determine physiological profiles including; pH, hematocrit, glucose, electrolytes, lactate, pCO2 (mmHg), SO2 (mmHg), and bicarbonate. Results showed increases in lactate and hematocrit immediately after surgical or simulated castration with decreases in pH, HCO3− and base excess (BE). Surgical castration produced marked alterations of the physiological profile, detected by reduced pH and HCO3, higher lactate levels and BE alterations. These changes indicated metabolic acidosis that was greater in the piglets castrated surgically with one horizontal incision than in those castrated with two vertical incisions. More research is needed on the use of lidocaine during surgical castration, as it showed no effect on physiological profile in this study, but did alter hematocrit values.

Reduced volume for direct PCR amplification of blood reference samples using Identifiler® Direct and Globalfiler™ Express assays

FTA® cards were developed for analysing reference samples such as blood or saliva and helping to store the sample for long term purposes as it kills the microorganisms that comes along with biological stains which can be potential inhibitors. The aim of this research is to optimize a reduced volume PCR amplification for STR analysis of reference blood samples. This approach will help to reduce the costs of DNA profiling using direct amplification multiplex assays. A total number of 130 blood samples from different individuals were collected on FTA® cards. The reaction was optimized using half volume for amplification with Identifiler® Direct and Globalfiler™ Express multiplex assays. The protocol was applied in a forensic laboratory and was able to reduce costs and account for variability of DNA amounts among reference samples.

Estimation of the binding potential BPND without a reference region or blood samples for brain PET studies

Binding potential (BPND) is a commonly used PET outcome measure because it can be estimated without blood sampling if a brain reference region (RR) devoid of the target of interest exists. For many radioligands, however, no RR exists, and the total distribution volume (VT), whose estimation requires arterial blood sampling, is normally considered as the outcome measure. Here, we present a method that allows calculation of BPND without requiring either blood samples or a RR.The method extends our previous algorithm for estimating non-displaceable distribution volumes (VND) without using a RR. Here we show that if a template input function, with arbitrary amplitude but a shape similar to the actual arterial input function, is used in the algorithm, estimation of VT and VND are both proportionally biased, and thus this bias cancels out in the estimation of BPND. The method is evaluated using simulated data, human data acquired with the serotonin 1A receptor radioligand [11C]WAY-100635, and blocking data acquired in baboons using the serotonin 1A receptor radioligand [11C]CUMI-101.We evaluated two versions of template input functions: an arbitrarily downscaled version of the actual arterial input function, and an unscaled population-based input function. In addition, we evaluated how shape modifications of the template input function impact the estimates of BPND.With the downscaled input function, BPND values close to the gold standard were obtained. When the unscaled population-based based input function was used, greater variability was observed but no discernable bias was introduced. When the input function shape was modified, a systematic but small bias in BPND was introduced.We conclude that, provided the shape of the arterial input function is adequately described, determination of its amplitude is not necessary for estimation of BPND.

Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition

ABSTRACTEpigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

Estimation and Correction of the Blood Volume Variations of Dried Blood Spots Using a Postcolumn Infused-Internal Standard Strategy with LC-Electrospray Ionization-MS.

Dried blood spots (DBSs) have had a long history in disease screening in newborns but have gained attention in recent years in the medical care of adults because of the growing importance of personalized medicine. DBSs have several advantages, such as easy transportation, cost-effectiveness, and minimally invasive biological sampling. There are two strategies to process DBS samples. One method takes a fixed diameter of subsample, and another requires the extraction of the whole spot. The whole-spot extraction method is less affected by hematocrit-caused errors, but it requires calibration of the blood volume. We propose a novel strategy using a postcolumn infused-internal standard (PCI-IS) method with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for estimating and correcting blood volume variations on the DBS cards. By using PCI-IS to measure the extent of ion suppression in the first ion suppression zone in the chromatogram, the blood volume on the DBS cards can be calculated and further calibrated. We used reference blood samples with different volumes (5 to 25 μL) to construct a calibration curve between the blood volume and the extent of ion suppression. The calibration curve was used to estimate the blood volume on the DBS cards collected from 6 volunteers, with 5 designated volumes from each volunteer. The estimation accuracy of the PCI-IS method was between 74.5% and 120.3%. The validated PCI-IS method was used to estimate and calibrate blood volume variation and also to quantify the voriconazole concentration for 26 patients undergoing voriconazole therapy. A high correlation was found for the quantification results between the DBS samples and the conventionally used plasma samples (r = 0.97). The PCI-IS method was demonstrated to be a simple and accurate method for estimating and calibrating the blood volume variation on DBS cards, which greatly facilitates using the DBS method for therapeutic drug monitoring (TDM) for improving the efficacy and safety of drug therapy.

Pharmacokinetic evaluation of cefdinir-loaded floating alginate beads in rabbits using LC–MS/MS

The present investigation aims to compare the pharmacokinetic parameters of Cefdinir in rabbits, from floating alginate (an anionic polysaccharide obtained from cell walls of brown algae) beads and conventional suspension, using a new LC–MS/MS method. Formulations equivalent to 20 mg/kg were administered orally to test and reference group and blood samples were collected at selected time intervals up to 24 h. Plasma concentrations of Cefdinir were determined using validated LC–MS/MS method and pharmacokinetic parameters were derived by non-compartment model. Statistically significant (p < 0.05) increase in Cmax, Tmax, AUC0–∞ and MRT was observed in case of floating alginate beads, whereas KE and t1/2 remained relatively constant. MRT and tmax increased significantly as a result of controlled drug release. Relative bioavailability was 337.45 % for the floating beads. Thus, alginate based floating formulation improve the bioavailability (3.37 fold) of Cefdinir compared to suspension. The absorption of Cefdinir from floating beads was found mainly from duodenum (73.0 %) and Jejunum 1 and 2 (13.0 %).