Components In Blood Samples Research Articles

Performance of IS6110-LAMP assay for detection of Mycobacterium tuberculosis complex in blood and urine samples from patients with extrapulmonary tuberculosis

BackgroundThe diagnosis of extrapulmonary tuberculosis (EPTB) shows numerous difficulties because of non-specific symptomatology and low sensitivity of conventional methods. Loop-mediated isothermal amplification (LAMP) is a fast and low-cost technique, which can amplify under isothermal conditions an amount of target DNA copies into approximately a billion copies. ObjectiveThe present study aimed to evaluate a IS6110-LAMP system for Mycobacterium tuberculosis detection in blood and urine samples from patients with EPTB. MethodsThe collected samples (n = 122) were stratified in two groups: Group EPTB – patient samples with confirmed EPTB (n = 61); Group non-TB – patient samples without TB (n = 61). The urine samples underwent decontamination, and the components of blood samples were separated (plasma and PBMC). DNA extractions were performed in all biological samples followed by IS6110-LAMP assay technique. The detection limit was evaluated through dilution curves (1:10) using Mtb reference strain (H37Rv) genomic DNA. FindingsThe detection limit of IS6110-LAMP was 10 fg/μL (∼10–20 bacilli/μL). The IS6110-LAMP technique sensitivity and specificity were 95.65 % and 79.25 %, respectively, with a general kappa agreement index of 0.762. Main conclusionsBased on these results, IS6110-LAMP test showed considerable diagnostic parameters, being able to aid in the speed and accuracy of the final EPTB diagnosis.

An Isothermal Molecular Point of Care Testing for African Swine Fever Virus Using Recombinase-Aided Amplification and Lateral Flow Assay Without the Need to Extract Nucleic Acids in Blood

African swine fever (ASF) is a highly contagious and usually deadly porcine infectious disease listed as a notifiable disease by the World Organization for Animal Health (OIE). It has brought huge economic losses worldwide, especially since 2018, the first outbreak in China. As there are still no effective vaccines available to date, diagnosis of ASF is essential for its surveillance and control, especially in areas far from city with limited resources and poor settings. In this study, a sensitive, specific, rapid, and simple molecular point of care testing for African swine fever virus (ASFV) B646L gene in blood samples was established, including treatment of blood samples with simple dilution and boiling for 5 min, isothermal amplification with recombinase-aided amplification (RAA) at 37°C in a water bath for 10 min, and visual readout with lateral flow assay (LFA) at room temperature for 10–15 min. Without the need to extract viral DNA in blood samples, the intact workflow from sampling to final diagnostic decision can be completed with minimal equipment requirement in 30 min. The detection limit of RAA-LFA for synthesized B646L gene-containing plasmid was 10 copies/μl, which was 10-fold more sensitive than OIE-recommended PCR and quantitative PCR. In addition, no positive readout of RAA-LFA was observed in testing classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, pseudorabies virus and porcine circovirus 2, exhibiting good specificity. Evaluation of clinical blood samples of RAA-LFA showed 100% coincident rate with OIE-recommended PCR, in testing both extracted DNAs and treated bloods. We also found that some components in blood samples greatly inhibited PCR performance, but had little effect on RAA. Inhibitory effect can be eliminated when blood was diluted at least 32–64-fold for direct PCR, while only a 2–4 fold dilution of blood was suitable for direct RAA, indicating RAA is a better choice than PCR when blood is used as detecting sample. Taken together, we established an sensitive, specific, rapid, and simple RAA-LFA for ASFV molecular detection without the need to extract viral DNA, providing a good choice for point of care testing of ASF diagnosis in the future.

Parallel-Coupled Dual SiOxNy Racetrack Resonators as Biosensors with High Improved Intrinsic Limit of Detection

This study describes a chip-integrated optical biosensor based on dual coupled racetrack resonators. A physical analysis of the device is carried out using conformal transformation and coupled-mode theory. The intrinsic limit of detection is improved by a factor of 100, compared to a conventional single-resonator system, with better dynamic range as well. The proposed sensor could be a promising candidate for analyzing different components of blood samples in medical diagnostics, which is especially important for the early detection of critical cancers, such as prostate cancer.

Ultrasmall gold nanorods: synthesis and glycocalyx-related permeability in human endothelial cells.

BackgroundClinical data show shed endothelial glycocalyx (GCX) components in blood samples of atherosclerotic patients, linking atherosclerotic development to endothelial GCX integrity. Healthy GCX has pores no >7 nm, and shed GCX has even larger pores. Therefore, we suggest targeting and treating atherosclerosis-prone blood vessels by using nanoscale vehicles to deliver drugs via the nanoscale GCX as it becomes dysfunctional.Materials and methodsTo test our idea, we investigated permeability of nanoparticles in endothelium, as related to a GCX expression. The present work involves nanorods, which are expected to interact with larger portions of endothelial cell (EC) membranes, due to surface area of the nanorod long axis. Conventional nanorod diameters are orders of magnitude larger than the GCX pore size, so we adapted conventional synthesis methods to fabricate ultrasmall gold nanorods (GNRs). Our ultrasmall GNRs have an aspect ratio of 3.4, with a length of 27.9±3.1 nm and a diameter of 8.2±1.4 nm. In addition, we produced GNRs that are biocompatible and fluorescently visible, by coating the surface with functionalized polyethylene glycol and Alexa Fluor 647. To study GNR–GCX interactions, we used human ECs, for species relevance.ResultsUnder life-like flow conditions, the human ECs are densely covered with a 1.3 µm thick layer of GCX, which coincides with minimal GNR permeability. When the GCX is weakened from lack of flow (static culture) or the presence of GCX degradation enzyme in the flow stream, the GCX shows 40% and 60% decreased thickness, respectively. GCX weakness due to lack of flow only slightly increases cellular permeability to GNRs, while GCX weakness due to the presence of enzyme in the flow leads to substantial increase in GNR permeability.ConclusionThese results clarify that the GCX structure is an avenue through which drug-carrying nanoparticles can be delivered for targeting affected blood vessels to treat atherosclerosis.

Stability of Biochemical Components in Blood Samples Transported by Tempus600/Sysmex GLP Robot Reception System.

The Department of Clinical Immunology and Biochemistry, Lillebaelt Hospital, Denmark, and TIMEDICO invented and developed the Tempus600®, a pneumatic tube system (PTS). This system is only 2.5 cm in diameter and sends blood tubes directly, without packing before transport. Request forms need not be enclosed, since samples are labeled with patient and barcode ID. At the end of the pipes, a robot receives and places the tubes onto a GLP transport system after the principle of "first in, first out" (FIFO), whereafter the tubes are delivered to centrifuges and analytical equipment. The whole system has decreased the total turnaround time (ToTAT) from 2 to 3 h to <60 min. The aim of the study was to validate the Tempus600/GLP robot system for the 89 most frequent or critical components. Two sets of blood samples were drawn from 100 patients. One set was sent by the Tempus600/GLP robot system, and the other was couriered directly to the laboratory for analysis. Results were compared using difference plots. The results for 85 of the components did not deviate from the optimized fast courier reference transport more than could be expected by the analytical variation. O2 saturation, oxyhemoglobin, and partial pressure of oxygen (Po2) deviated considerably from a clinical point of view. Partial pressure of carbon dioxide (Pco2) results were clinically acceptable for assessing patients with obstructive lung disease for hypercapnia. The Tempus600/GLP robot system can be used for transport of the majority of routinely used analytical tests.

Telling biological time from a blood sample: current capabilities and future potential.

Circadian rhythms, near-24 h oscillations that reflect homeostatic control by an internal timing system rather than the influence of external factors, are an important and sometimes underappreciated aspect of human physiology and biochemistry. Over the past few decades, the pineal gland hormone melatonin has been established both as a robust marker of circadian phase in plasma or saliva and as a chronobiotic drug administered to reset the timing of the circadian oscillator. Recent work by our own and other laboratories has sought to systematically investigate whole categories of molecular components in blood samples in a hypothesis-free fashion by employing metabolomic methodologies to study low molecular weight compounds and transcriptomic methodologies to study gene expression in white blood cells, respectively. A number of components have been pinpointed that show a rhythmic circadian variation or are affected by imposed factors such as sleep deprivation. Although melatonin, a robust and reliable circadian phase marker, will be a hard act to follow, these lines of research suggest numerous potential leads for useful new markers of biological timing.

Rapid Analysis of Dried Blood Spot Samples with Sub-2-µM LC–MS/MS

The use of dried blood and dried plasma spots for storage and transportation of samples derived from clinical trials holds the promise to reduce cost, simplify storage and shipping as well as reducing animal usage. From the bioanalysts' point of view, these dried-paper samples add an extra layer of complexity to the analysis introducing extra matrix effects from the paper itself and sometimes from antiviral treatments applied to the card. In this article we demonstrate the use of the sub-2-µm particle LC-MS/MS for the bioanalysis of samples derived from a dried blood spot. The higher resolution provided by these small-particle separations allowed for greater resolution of the analyte from the endogenous components in blood samples and from the card-treatment chemicals. The method-development process was enhanced by the use of MS, which could simultaneously acquire full scan and multiple reaction monitoring data, allowing resolution from metabolites and endogenous matrix components. The use of this approach produced sensitivity levels in the 50-100 pg/ml range and analysis times in the 1-2 min range, which was five-times more sensitive and three-times faster than HPLC. This throughput and sensitivity makes this approach ideal for the analysis of preclinical and clinical studies derived from dried blood spots.

Alternative complement pathway deregulation is correlated with dengue severity.

BackgroundThe complement system, a key component that links the innate and adaptive immune responses, has three pathways: the classical, lectin, and alternative pathways. In the present study, we have analyzed the levels of various complement components in blood samples from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients and found that the level of complement activation is associated with disease severity.Methods and ResultsPatients with DHF had lower levels of complement factor 3 (C3; p = 0.002) and increased levels of C3a, C4a and C5a (p<0.0001) when compared to those with the less severe form, DF. There were no significant differences between DF and DHF patients in the levels of C1q, immunocomplexes (CIC-CIq) and CRP. However, small but statistically significant differences were detected in the levels of MBL. In contrast, the levels of two regulatory proteins of the alternative pathway varied widely between DF and DHF patients: DHF patients had higher levels of factor D (p = 0.01), which cleaves factor B to yield the active (C3bBb) C3 convertase, and lower levels of factor H (p = 0.03), which inactivates the (C3bBb) C3 convertase, than did DF patients. When we considered the levels of factors D and H together as an indicator of (C3bBb) C3 convertase regulation, we found that the plasma levels of these regulatory proteins in DHF patients favored the formation of the (C3bBb) C3 convertase, whereas its formation was inhibited in DF patients (p<0.0001).ConclusionThe data suggest that an imbalance in the levels of regulatory factors D and H is associated with an abnormal regulation of complement activity in DHF patients.

Machine recognition in medical microscopy.

Some general aspects of machine recognition are discussed. The advantages and limitations of machine systems are compared with those of trained human observers and the problems involved in introducing machines into classification systems designed for human observers are outlined. The present classification system for the cellular components of blood samples is described, with emphasis on the different types of white cell encountered. Cervical samples and chromosomes are described and discussed from the machine recognition point of view. All proposed machine recognition systems in microscopy must satisfy certain technical criteria; these are listed and discussed briefly. A number of earlier attempts to recognise cells are described; the results obtained and the difficulties encountered are outlined. The work of a number of university, medical and industrial research groups actively engaged in this field is described. The likely developments in this field are discussed from the point of view of improvements which may occur in the hardware of scanning and computing and also the better understanding, integration and exploration of machine systems in medical research and in routine medical diagnostic procedures.