Unprocessed Blood Samples Research Articles

A Bio-Inspired Free-Standing Film with Versatility: From Heterogeneous CTCs Programed Isolating to Breast Cancer Molecular Typing.

Developing a robust strategy for profiling heterogeneous circular tumor cells specifically, distinguishing the phenotypes of which in blood sample of cancer patient precisely, and releasing them sequentially, is significant for cancer management by liquid biopsy. Herein, a bio-inspired free-standing and flexible film composed of TiO2 nanotube and silk fibroin, fabricated with multiply dynamic bioactive surface (TSF/MDBS) by a simple and eco-friendly way including using polydopamine chemistry and dual dynamic covalent chemistry, is reported. The as-prepared TSF/MDBS binds specific peptides toward cells with epithelial biomarker and human epithelial growth factor receptor 2 (HER2) biomarker, and antifouling agents bovine serum albumin for obviating platelets and proteins adhering of blood, can capture heterogeneous CTCs with enhanced capability due to the cytocompatible soft film and exquisite surface design, and further release the captured cells as program, by specifically breaking down the covalent bonds in sequence via the action of adding biocompatible molecules fructose and glutathione. By applying the TSF/MDBS, it can be tailored into desired pieces for identifying CTCs with different phenotypes (HER2-high and HER2-low) from the unprocessed blood samples of breast cancer patients, and finally profiling these heterogeneous CTCs, to discriminate HER2 positive or negative of breast cancer patients in clinical applications.

Potential of Point-of-Care and At-Home Assessment of Immune Status via Rapid Cytokine Detection and Questionnaire-Based Anamnesis.

Monitoring the immune system’s status has emerged as an urgent demand in critical health conditions. The circulating cytokine levels in the blood reflect a thorough insight into the immune system status. Indeed, measuring one cytokine may deliver more information equivalent to detecting multiple diseases at a time. However, if the reported cytokine levels are interpreted with considering lifestyle and any comorbid health conditions for the individual, this will promote a more precise assessment of the immune status. Therefore, this study addresses the most recent advanced assays that deliver rapid, accurate measuring of the cytokine levels in human blood, focusing on add-on potentials for point-of-care (PoC) or personal at-home usage, and investigates existing health questionnaires as supportive assessment tools that collect all necessary information for the concrete analysis of the measured cytokine levels. We introduced a ten-dimensional featuring of cytokine measurement assays. We found 15 rapid cytokine assays with assay time less than 1 h; some could operate on unprocessed blood samples, while others are mature commercial products available in the market. In addition, we retrieved several health questionnaires that addressed various health conditions such as chronic diseases and psychological issues. Then, we present a machine learning-based solution to determine what makes the immune system fit. To this end, we discuss how to employ topic modeling for deriving the definition of immune fitness automatically from literature. Finally, we propose a prototype model to assess the fitness of the immune system through leveraging the derived definition of the immune fitness, the cytokine measurements delivered by a rapid PoC immunoassay, and the complementary information collected by the health questionnaire about other health factors. In conclusion, we discovered various advanced rapid cytokine detection technologies that are promising candidates for point-of-care or at-home usage; if paired with a health status questionnaire, the assessment of the immune system status becomes solid and we demonstrated potentials for promoting the assessment tool with data mining techniques.

Seven day pre-analytical stability of serum and plasma neurofilament light chain

Neurofilament light chain (NfL) has emerged as a biomarker of neuroaxonal damage in several neurologic conditions. With increasing availability of fourth-generation immunoassays detecting NfL in blood, aspects of pre-analytical stability of this biomarker remain unanswered. This study investigated NfL concentrations in serum and plasma samples of 32 patients with neurological diagnoses using state of the art Simoa technology. We tested the effect of delayed freezing of up to 7 days and statistically determined stability and validity of measured concentrations. We found concentrations of NfL in serum and plasma to remain stable at room temperature when processing of samples is delayed up to 7 days (serum: mean absolute difference 0.9 pg/mL, intraindividual variation 1.2%; plasma: mean absolute difference 0.5 pg/mL, intraindividual variation 1.3%). Consistency of these results was nearly perfect for serum and excellent for plasma (intraclass correlation coefficients 0.99 and 0.94, respectively). In conclusion, the soluble serum and plasma NfL concentration remains stable when unprocessed blood samples are stored up to 7 days at room temperature. This information is essential for ensuring reliable study protocols, for example, when shipment of fresh samples is needed.

Fe3O4@Au SERS tags-based lateral flow assay for simultaneous detection of serum amyloid A and C-reactive protein in unprocessed blood sample

Accurate detection of mutiple biomarkers in unprocessed blood via lateral flow immunoassay (LFA) strips remains challenging. Herein, we reported a novel surface-enhanced Raman scattering (SERS)-based LFA strip for ultrasensitive and simultaneous analysis of dual infection biomarkers by using functionalized Fe3O4@Au magnetic nanoparticles (MNPs). Antibody-modified Fe3O4@Au MNPs were designed as SERS nanotags and efficient enrichment tool. The target infection biomarkers were quickly captured and enriched from the blood sample by Fe3O4@Au nanotags. After washing to remove impurities, Fe3O4@Au nanotags acted as high-performance SERS reporters for quantitative detection of the target in the LFA strip. Through this strategy, the proposed Fe3O4@Au based LFA allowed quantified analysis of target infection biomarkers in unprocessed blood samples with the limits of detection of 0.1 ng/mL and 0.01 ng/mL for serum amyloid A (SAA) and C-reactive protein (CRP), respectively. By comparison, the proposed assay for SAA and CRP was 100-fold and 1000-fold more sensitive than standard colloidal gold strip by using the same antibodies, respectively. This magnetic SERS strip also exhibited excellent stability, specificity, and selectivity for complex samples and could be used as an effective tool for infectious disease diagnostics.

Whole genome sequencing of amplified Plasmodium knowlesi DNA from unprocessed blood reveals genetic exchange events between Malaysian Peninsular and Borneo subpopulations

The zoonotic Plasmodium knowlesi parasite is the most common cause of human malaria in Malaysia. Genetic analysis has shown that the parasites are divided into three subpopulations according to their geographic origin (Peninsular or Borneo) and, in Borneo, their macaque host (Macaca fascicularis or M. nemestrina). Whilst evidence suggests that genetic exchange events have occurred between the two Borneo subpopulations, the picture is unclear in less studied Peninsular strains. One difficulty is that P. knowlesi infected individuals tend to present with low parasitaemia leading to samples with insufficient DNA for whole genome sequencing. Here, using a parasite selective whole genome amplification approach on unprocessed blood samples, we were able to analyse recent genomes sourced from both Peninsular Malaysia and Borneo. The analysis provides evidence that recombination events are present in the Peninsular Malaysia parasite subpopulation, which have acquired fragments of the M. nemestrina associated subpopulation genotype, including the DBPβ and NBPXa erythrocyte invasion genes. The NBPXb invasion gene has also been exchanged within the macaque host-associated subpopulations of Malaysian Borneo. Our work provides strong evidence that exchange events are far more ubiquitous than expected and should be taken into consideration when studying the highly complex P. knowlesi population structure.

A microfluidic device for label-free isolation of tumor cell clusters from unprocessed blood samples.

Primary cancers disseminate both single circulating tumor cells (CTCs) and CTC "clusters," the latter of which have been shown to demonstrate greater metastatic propensity and adverse impact on prognosis. Many devices developed to isolate single CTCs also capture CTC clusters, but there is translational potential for a platform specifically designed to isolate CTC clusters. Herein, we introduce our microfluidic device for isolating CTC clusters ("Microfluidic Isolation of CTC Clusters" or MICC), which is equipped with ∼10 000 trap chambers that isolate tumor cell clusters based on their large sizes and dynamic force balance against a pillar obstacle in the trap chamber. Whole blood is injected, followed by a wash step to remove blood cells and a final backflush to release intact clusters for downstream analysis. Using clusters from tumor cell-line and confocal microscopy, we verified the ability of the MICC platform to specifically capture tumor cell clusters in the trap chambers. Our flow rate optimization experiments identified 25 μl/min for blood injection, 100 μl/min as wash flow rate, and 300 μl/min as the release flow rate - indicating that 1 ml of whole blood can be processed in less than an hour. Under these optimal flow conditions, we assessed the MICC platform's capture and release performance using blood samples spiked with different concentrations of clusters, revealing a capture efficiency of 66%-87% and release efficiency of 76%-90%. The results from our study suggest that the MICC platform has the potential to isolate CTC clusters from cancer patient blood, enabling it for clinical applications in cancer management.

A SERS nano-tag-based magnetic-separation strategy for highly sensitive immunoassay in unprocessed whole blood

Assay technologies capable of detecting biomarker concentrations in unprocessed whole blood samples are fundamental for applications in medical diagnostics. SERS nano-tags integrated magnetic-separation biosensor (MSB) was realized for the first time for immunoassay in whole blood. The reliability and sensitivity of this method rely, in a large extent, on the quality and properties of the SERS nano-tags. The constructed silicacoated Ag SERS nano-tags as labels were used in a rapid and specific MSB immune sensor to detect Matrix Metalloproteinases 9 (MMP-9) in unprocessed blood samples. With fast screening ability and outstanding sensitivity, we anticipate that this method would greatly promote practical application of stroke-based early-stage cancer diagnosis.

Immobilization of Proline Dehydrogenase on Functionalized Silica Mesoporous Nanomaterial Towards Preparation of a Novel Thermostable Enzyme Biosensor

In this report, a thermostable electrochemical biosensor for sensitive determination of L-proline in whole blood was prepared by immobilization of proline dehydrogenase within n-Propylamine functionalized mobile crystalline material-41 doped with iron oxide nanoparticles. By combining of magnetic mesoporous silica with proline dehydrogenase and using voltammetry analysis, a proline biosensor was fabricated on glassy carbon electrode pre-modified by poly cysteine supported beta cyclodextrin. The magnetic mesoporous silica networks having a high surface area (362 m2 g−1) exhibited excellent properties for entrapment of proline dehydrogenase. The prepared sensor was used as an electrochemical detector for selective detection of L-proline at physiological pH. The results show that the oxidation of L-proline is ir-reversible and controlled by diffusion. Using differential pulse voltammetry, the oxidation peak current was proportional to L-proline concentration in the range of 1–100 mM and 1 μM–1 nM, while the low limit of quantification was 1 nM under physiological pH. For proof-of-technology, the biosensor was used for the detection of L-proline in biological samples such as whole blood and human blood serum of neonates. It is demonstrated that engineered biosensor provides a rapid and sensitive method for determination of L-proline in neonatal blood serum samples. The current response of electrocatalytic oxidation of L-Pro was maintained in the analytical solution temperature up to 310 °K. The applied approach led to better resistance to temperature and pH inactivation in comparison to the free enzyme. The method is a potential tool for diagnostic of neonatal hyperprolinemia. Taking a good facet of the biosensor into account, it was implemented for the detection of the L-Pro in normal cell (L929) and various cancer cell lines such as gastric cancer cell-CAT 3, colon cancer cell-HCT, colon cancer cell-SW, and breast cancer cell-MCF7. Finally, this sensor was used to determine of L-Pro in unprocessed blood samples.

Nucleic acid hybridization on an electrically reconfigurable network of gold-coated magnetic nanoparticles enables microRNA detection in blood.

There is intense interest in quantifying the levels of microRNA because of its importance as a blood-borne biomarker. The challenge has been to develop methods that can monitor microRNA expression both over broad concentration ranges and in ultralow amounts directly in a patient's blood. Here, we show that, through electric-field-induced reconfiguration of a network of gold-coated magnetic nanoparticles modified by probe DNA (DNA-Au@MNPs), it is possible to create a highly sensitive sensor for direct analysis of nucleic acids in samples as complex as whole blood. The sensor is the first to be able to detect concentrations of microRNA from 10 aM to 1 nM in unprocessed blood samples. It can distinguish small variations in microRNA concentrations in blood samples of mice with growing tumours. The ultrasensitive and direct detection of microRNA using an electrically reconfigurable DNA-Au@MNPs network makes the reported device a promising tool for cancer diagnostics.

A SERS nano-tag-based fiber-optic strategy for in situ immunoassay in unprocessed whole blood

Assay technologies capable of detecting biomarker concentrations in unprocessed whole blood samples are fundamental for applications in medical diagnostics. SERS nano-tags integrated fiber-optic biosensor (FOB) was realized for the first time for in situ immunoassay in whole blood. The reliability and sensitivity of this method rely, in a large extent, on the quality and properties of the SERS nano-tags. The constructed silica-coated Ag SERS nano-tags as labels were used in a rapid and specific in situ FOB immune sensor to detect alpha fetoprotein (AFP) in unprocessed blood samples. Preliminary results of in vivo and in situ dynamic observation of AFP of whole blood in wistar rat highlight the power of this new method.

Stability of cytokines, chemokines and soluble activation markers in unprocessed blood stored under different conditions

BackgroundBiomarkers such as cytokines, chemokines, and soluble activation markers can be unstable when processing of blood is delayed. The stability of various biomarkers in serum and plasma was investigated when unprocessed blood samples were stored for up to 24h at room and refrigerator temperature. MethodsBlood was collected from 16 healthy volunteers. Unprocessed serum, EDTA and heparinized blood was stored at room (20–25°C) and refrigerator temperature (4–8°C) for 0.5, 2, 4, 6, 8, and 24h after collection before centrifugation and separation of serum and plasma. Samples were batch tested for various biomarkers using commercially available immunoassays. Statistically significant changes were determined using the generalized estimating equation. ResultsIFN-γ, sIL-2Rα, sTNF-RII and β2-microglobulin were stable in unprocessed serum, EDTA and heparinized blood samples stored at either room or refrigerator temperature for up to 24h. IL-6, TNF-α, MIP-1β and RANTES were unstable in heparinized blood at room temperature; TNF-α, and MIP-1β were unstable in unprocessed serum at room temperature; IL-12 was unstable in unprocessed serum at refrigerator temperature; and neopterin was unstable in unprocessed EDTA blood at room temperature. IL-1ra was stable only in unprocessed serum at room temperature. ConclusionAll the biomarkers studied, with the exception of IL-1ra, were stable in unprocessed EDTA blood stored at refrigerator temperature for 24h. This indicates that blood for these biomarkers should be collected in EDTA and if delays in processing are anticipated the unseparated blood should be stored at refrigerator temperature until processing.

Impact of Blood Sample Collection and Processing Methods on Glucose Levels in Community Outreach Studies

Glucose obtained from unprocessed blood samples can decrease by 5%–7% per hour due to glycolysis. This study compared the impact of glucose degradation on measured glucose values by examining two different collection methods. For the first method, blood samples were collected in tubes containing sodium fluoride (NaF), a glycolysis inhibitor. For the second method, blood samples were collected in tubes containing a clot activator and serum gel separator and were centrifuged to separate the serum and plasma 20 minutes after sample collection. The samples used in the two methods were collected during the same blood draw and were assayed by the clinical laboratory 2–4 hours after the samples were obtained. A total of 256 pairs of samples were analyzed. The average glucose reading for the centrifuged tubes was significantly higher than the NaF tubes by 0.196 ± 0.159 mmol/L (P < 0.01) or 4.2%. This study demonstrates the important role collection methods play in accurately assessing glucose levels of blood samples collected in the field, where working environment may be suboptimal. Therefore, blood samples collected in the field should be promptly centrifuged before being transported to clinical labs to ensure accurate glucose level measurements.