Blood Fractionation Research Articles

In-vitro blood purification using tiny pinch holographic optical tweezers based on deep learning

In-vitro blood purification is essential to a wide range of medical treatments, requiring fine-grained analysis and precise separation of blood components. Despite existing methods that can extract specific components from blood by size or by magnetism, there is not yet a general approach to efficiently filter blood components on demand. In this work, we introduce the first programmable non-contact blood purification system for accurate blood component detection and extraction. To accurately identify different cells and artificial particles in the blood, we collected and annotated a new blood component object detection dataset and trained a collection of deep-learning-based object detectors upon it. To precisely capture and extract desired blood components, we fabricated a microfluidic chip and set up a customized holographic optical tweezer to trap and move cells/particles in the blood. Empirically, we demonstrate that our proposed system can perform real-time blood fractionation with high precision reaching up to 96.89%, as well as high efficiency. Its scalability and flexibility open new research directions in blood treatment.

Abstract 5018: Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications

Abstract “For research use only. Not for diagnostic procedures.” Introduction: Liquid biopsy is a non-invasive alternative to tissue biopsies for cancer profiling using cell-free total nucleic acids (cfTNA; including cell free DNA (cfDNA) and cell free RNA (cfRNA)), the biological analytes derived from tumor cells floating in blood. However, it is crucial to optimize the whole blood fractionation workflows, since limited access to a suitable centrifuge at blood collection sites poses a challenge to this critical fractionation step, causing delays in processing the blood, leading to decreased sensitivity of variant detection and increased occurrence of false positives. Current study shows a systematic comparison of the effects of 8 different blood fractionation conditions for spin speeds, temperatures and duration on the levels of cfTNA derived from K2-EDTA-derived blood from multiple healthy and non-small cell lung cancer (NSCLC) donors, using next generation sequencing (NGS). Methods: CfTNA from K2-EDTA derived plasma was isolated using the MagMAXTM Cell-Free Total Nucleic Acid kit and the Genexus™ Cell free total nucleic acid kit, utilizing the KingFisher TM Flex and GenexusTMpurification systems. Yield and integrity of cfTNA isolated was evaluated by Qubit™, Agilent Tapestation and quantitative real time PCR (qRT-PCR). Furthermore, the impact of these test conditions on quality control specifications of DNA and RNA was assessed by performing sequencing on an Ion TorrentTM GenexusTM sequencing instrument using the AmpliSeqTM HD target amplification assay, that reports sequence variations in DNA for 42 cancer related genes and RNA in 7 cancer related genes. Results: Results showed comparable performance for all the 8 blood fractionation conditions tested, for yield and cfDNA profiles compared to control condition, indicating that there is no significant impact of these conditions on either yield or integrity of the cfDNA isolated. In addition, NGS data also showed that on target reads and median molecular coverage for DNA are comparable for all the conditions tested. However, sequencing data demonstrated that one test condition showed significantly better performance for RNA quality control specifications in terms of RNA mapped reads and molecular coverage for process controls used in the assay, compared to all other conditions tested. Conclusion: Data suggests that one of the whole blood fractionation conditions tested impact the cfRNA recovery from plasma without negatively affecting the yield, and integrity of isolated cfDNA and shows increased sensitivity for RNA process controls in NGS. Citation Format: MADHU JASTI, Sergio Hernandez, Nick Siepert, Lokesh Kodandaramaswamy, Thilanka Jayaweera, Jeoffrey Schageman, Angie Cheng, Susanne Lopez, Miao Der Chen. Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5018.

Abstract 5030: Evaluation of different blood collection tubes for liquid biopsy NGS applications

Abstract Intro: Liquid biopsy is fast becoming a standard practice in oncology research due to recent advancements in Next Generation Sequencing (NGS). To detect genetic variants, whole blood samples are collected in blood collection tubes (BCTs) and fractionated to obtain plasma which is then used to isolate cell free nucleic acid (cfNA) for analysis. While the limited availability of cfNA is a challenge in liquid biopsy, storage condition and duration between blood collection and fractionation have a significant impact on the quantity and quality of the cfNA due to degradation and genomic DNA and RNA contamination. There are several BCTs available on the market offering stabilization of cfNA profiles to overcome the above challenges. Methods: Here we compare the NGS performance of cfNA derived from three commonly used BCTs on the market, BD Vacutainer™ K2EDTA Tubes, Streck™ Cell-Free DNA, and PAXgene® Blood ccfDNA tubes. Matched whole blood samples collected in the above-mentioned tube types were obtained from commercial vendors which were derived from 38 healthy donors as well as 6 cancer research samples. Whole blood samples were fractionated as per manufacturer recommendations and cfNA was isolated and quantified using Genexus™ Cell-Free Total Nucleic Acid Purification Kit on Genexus™ purification system. Isolated cfNA was then sequenced using Ion Ampliseq™ HD target amplification assay on Genexus™ Integrated sequencer. Results: Data shows that K2EDTA collected samples contain slightly higher amounts of cfNA compared to PAXgene® and Streck™ samples. When the cfDNA profiles were analyzed by Agilent™ BioAnalyzer™, K2EDTA derived cfDNA showed varying degrees of genomic DNA contamination which can inflate the cfDNA concentration. However, both Streck™ and PAXgene® derived cfDNA showed little to no detectable genomic DNA contamination. As a result, a higher percentage of PAXgene® and Streck™ samples did not meet the minimum input requirement (5ng) for the sequencing assay. Sequencing data for DNA showed that K2EDTA samples had higher total reads and median molecular coverage compared to the other two BCT types. However, all samples had passing QC metrics, and mean read length, on target reads, and uniformity were comparable between the three tube types. When DNA variant detection was evaluated using cancer research samples, no significant difference was observed in performance between K2EDTA and PAXgene® samples. Interestingly, sequencing data for RNA shows that PAXgene® samples had slightly higher mapped reads on average, and significantly higher internal process control coverage compared to both K2EDTA and Streck™ samples. Whereas Streck™ samples had significantly lower internal process control coverage. Conclusion: In summary, this study demonstrates the advantages and disadvantages of using different BCTs for liquid biopsy applications, in terms of cfNA yields, isolated DNA profiles, and sequencing performance. Citation Format: Nicholas Siepert, Emilia Ostrowska, Luming Qu He, Madhuri Jasti, Kris Lea, Thilanka Jayaweera, Angie Cheng. Evaluation of different blood collection tubes for liquid biopsy NGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5030.

Blood component separation in straight microfluidic channels.

Separation of blood components is required in many diagnostic applications and blood processes. In laboratories, blood is usually fractionated by manual operation involving a bulk centrifugation equipment, which significantly increases logistic burden. Blood sample processing in the field and resource-limited settings cannot be readily implemented without the use of microfluidic technology. In this study, we developed a small footprint, rapid, and passive microfluidic channel device that relied on margination and inertial focusing effects for blood component separation. No blood dilution, lysis, or labeling step was needed as to preserve sample integrity. One main innovation of this work was the insertion of fluidic restrictors at outlet ports to divert the separation interface into designated outlet channels. Thus, separation efficiency was significantly improved in comparison to previous works. We demonstrated different operation modes ranging from platelet or plasma extraction from human whole blood to platelet concentration from platelet-rich plasma through the manipulation of outlet port fluidic resistance. Using straight microfluidic channels with a high aspect ratio rectangular cross section, we demonstrated 95.4% platelet purity extracted from human whole blood. In plasma extraction, 99.9% RBC removal rate was achieved. We also demonstrated 2.6× concentration of platelet-rich plasma solution to produce platelet concentrate. The extraction efficiency and throughput rate are scalable with continuous and clog-free recirculation operation, in contrast to other blood fractionation approaches using filtration membranes or affinity-based purification methods. Our microfluidic blood separation method is highly tunable and versatile, and easy to be integrated into multi-step blood processing and advanced sample preparation workflows.

Blood-derived biomaterials for tissue graft biofabrication by solvent-based extrusion bioprinting

This article provides an overview of the different types of blood-derived biomaterials that can be used as solvent additives in the formulation of inks/bioinks for use in solvent extrusion printing/bioprinting. We discuss the properties of various blood sub-products obtained after blood fractionation in terms of their use in tailoring ink/bioink to produce functional constructs designed to improve tissue repair. Blood-derived additives include platelets and/or their secretome, including signaling proteins and microvesicles, which can drive cell migration, inflammation, angiogenesis, and synthesis of extracellular matrix proteins. The contribution of plasma to ink/bioink functionalization relies not only on growth factors, such as hepatocyte growth factor and insulin growth factors, but also on adhesive proteins, such as fibrinogen/fibrin, vitronectin, and fibronectin. We review the current developments and progress in solvent-based extrusion printing/bioprinting with inks/bioinks functionalized with different blood-derived products, leading toward the development of more advanced patient-specific 3D constructs in multiple medical fields, including but not limited to oral tissues and cartilage, bone, skin, liver, and neural tissues. This information will assist researchers in identifying the most suitable blood-derived product for their ink/bioink formulation based on the intended regenerative functionality of the target tissue.

Label-free microfluidic cell sorting and detection for rapid blood analysis.

Blood tests are considered as standard clinical procedures to screen for markers of diseases and health conditions. However, the complex cellular background (>99.9% RBCs) and biomolecular composition often pose significant technical challenges for accurate blood analysis. An emerging approach for point-of-care blood diagnostics is utilizing "label-free" microfluidic technologies that rely on intrinsic cell properties for blood fractionation and disease detection without any antibody binding. A growing body of clinical evidence has also reported that cellular dysfunction and their biophysical phenotypes are complementary to standard hematoanalyzer analysis (complete blood count) and can provide a more comprehensive health profiling. In this review, we will summarize recent advances in microfluidic label-free separation of different blood cell components including circulating tumor cells, leukocytes, platelets and nanoscale extracellular vesicles. Label-free single cell analysis of intrinsic cell morphology, spectrochemical properties, dielectric parameters and biophysical characteristics as novel blood-based biomarkers will also be presented. Next, we will highlight research efforts that combine label-free microfluidics with machine learning approaches to enhance detection sensitivity and specificity in clinical studies, as well as innovative microfluidic solutions which are capable of fully integrated and label-free blood cell sorting and analysis. Lastly, we will envisage the current challenges and future outlook of label-free microfluidics platforms for high throughput multi-dimensional blood cell analysis to identify non-traditional circulating biomarkers for clinical diagnostics.

Experimental Characterization of a Microfluidic Device Based on Passive Crossflow Filters for Blood Fractionation

The separation of red blood cells (RBCs) from blood plasma and the analysis of individual RBCs are of great importance, as they provide valuable information regarding the health of their donor. Recent developments in microfluidics and microfabrication have contributed to the fabrication of microsystems with complex features to promote the separation and analysis of RBCs. In this work, the separation capacity of a multi-step crossflow microfluidic device was evaluated by using a blood analogue fluid made by Brij L4 micelles and human RBCs separated from whole blood, suspended in a solution with hematocrits (Ht) of 0.5 and 1%. All the samples collected at the outlets of the device were experimentally analyzed and compared. The absorbance spectrum was also measured for the prepared blood samples. The results indicate that the tested blood analogue fluid has exhibited a flow behavior similar to that of blood. In addition, the optical absorbance spectrophotometry revealed that it was possible to evaluate the separation efficiency of the microfluidic device, concluding that the concentration of cells was lower at the most lateral outside outlets of the microchannel due to the cumulative effect of the multiple cross-flow filters.

Multiplexed micronutrient, inflammation, and malarial antigenemia assessment using a plasma fractionation device.

Processing and storing blood samples for future analysis of biomarkers can be challenging in resource limited environments. The preparation of dried blood spots (DBS) from finger-stick collection of whole blood is a widely used and established method as DBS are biosafe, and allow simpler field processing, storage, and transport protocols than serum or plasma. Therefore, DBS are commonly used in population surveys to assess infectious disease and/or micronutrient status. Recently, we reported that DBS can be used with the Q-plex™ Human Micronutrient 7-plex Array (MN 7-plex), a multiplexed immunoassay. This tool can simultaneously quantify seven protein biomarkers related to micronutrient deficiencies (iodine, iron and vitamin A), inflammation, and malarial antigenemia using plasma or serum. Serum ferritin, an iron biomarker, cannot be measured from DBS due to red blood cell (RBC) ferritin content confounding the results. In this study, we assess a simple blood fractionation tool that passively separates plasma from other blood components via diffusion through a membrane into a plasma collection disc (PCD). We evaluated the concordance of MN 7-plex analyte concentrations from matched panels of eighty-eight samples of PCD, DBS, and wet plasma prepared from anticoagulated venous whole blood. The results showed good correlations of >0.93 between the eluates from PCD and DBS for each analyte except ferritin; while correlations seen for plasma/PCD were weaker. However, the recovery rate of the analytes from the PCD were better than those from DBS. The serum ferritin measures from the PCD were highly correlated to wet plasma samples (0.85). This suggests that surveillance for iron status in low resource settings can be improved over the current methods restricted to only measuring sTfR in DBS. When used in combination with the MN 7-plex, all seven biomarkers can be simultaneously measured using eluates from the PCDs.

Experimental setup for the ultrasonic fractionation of flowing whole blood in a capillary

Abstract If a long ultrasound driving pulse is chosen such, that its frequency is greater than the resonance frequency of one cell type and less than the resonance frequency of another cell type, the cells are separated in different nodes and antinodes of the standing sound field. The purpose of this study was to build an experimental setup for the ultrasonic fractionation of whole blood in flow conditions. Blood fractionation is the separation of whole blood into its separate components, commonly used for analysis and transfusions. The experimental setup comprised a container with singleelement ultrasound transducers customisable in elevation and azimuth, focussed on a capillary through which blood was flowing. The acoustic focus coincided with the optical focus of a microscopic system to which a high-speed camera was attached. Ultrasound transducers were used with centre frequencies between 1MHz and 20MHz and with pressure amplitudes less than 1MPa. The duty cycle was greater than 10% in all experiments. The field of view was 0.2×0.2mm2. The optical resolution and camera speed allowed for visibility of red and white blood cells during flow conditions. Preliminary results show that red and white blood cells could be forced to move in opposite directions. The acoustic frequencies and pressures used were representative for those used in clinical diagnosis. We have shown that ultrasonic whole blood fractionation is feasible using ultrasound. Sonic fractionation speed might be an indicator for diseases such as malaria.

Higher Radiation Dose to the Immune Cells Correlates with Worse Tumor Control and Overall Survival in Patients with Stage III NSCLC: A Secondary Analysis of RTOG0617.

Simple SummaryEmerging evidence indicates that the immune system plays an important role in controlling tumors during radiotherapy, and radiation-induced immune toxicity such as lymphopenia is associated with poor survival. However, the immune system is not considered as a critical organ at risk in radiotherapy partially because the radiation dose to the immune system is difficult to compute. In this study, we developed a model to compute the radiation dose to the circulating blood, which contains the majority of active immune cells. We then validated this model by examining the correlations of the blood dose with treatment outcome for patients enrolled in the NRG/RTOG0617 phase III clinical trial. We demonstrated that the blood dose was significantly and independently associated with overall survival and local progression-free survival. This result suggests that radiation dose to circulating immune cells is critical for tumor control, and decreasing the dose to the immune system has the potential to improve survival.Background: We hypothesized that the Effective radiation Dose to the Immune Cells (EDIC) in circulating blood is a significant factor for the treatment outcome in patients with locally advanced non-small-cell lung cancer (NSCLC). Methods: This is a secondary study of a phase III trial, NRG/RTOG 0617, in patients with stage III NSCLC treated with radiation-based treatment. The EDIC was computed as equivalent uniform dose to the entire blood based on radiation doses to all blood-containing organs, with consideration of blood flow and fractionation effect. The primary endpoint was overall survival (OS), and the secondary endpoints were progression-free survival (PFS) and local progression-free survival (LPFS). The EDIC–survival relationship was analyzed with consideration of clinical significant factors. Results: A total of 456 patients were eligible. The median EDIC values were 5.6 Gy (range, 2.1–12.2 Gy) and 6.3 Gy (2.1–11.6 Gy) for the low- and high-dose groups, respectively. The EDIC was significantly associated with OS (hazard ratio [HR] = 1.12, p = 0.005) and LPFS (HR = 1.09, p = 0.02) but PFS (HR = 1.05, p = 0.17) after adjustment for tumor dose, gross tumor volume and other factors. OS decreased with an increasing EDIC in a non-linear pattern: the two-year OS decreased first with a slope of 8%/Gy when the EDIC < 6 Gy, remained relatively unchanged when the EDIC was 6–8 Gy, and followed by a further reduction with a slope of 12%/Gy when the EDIC > 8 Gy. Conclusions: The EDIC is a significant independent risk factor for poor OS and LPFS in RTOG 0617 patients with stage III NSCLC, suggesting that radiation dose to circulating immune cells is critical for tumor control. Organ at risk for the immune system should be considered during RT plan.

The Story of Blood for Shock Resuscitation: How the Pendulum Swings

Whole blood transfusion (WBT) began in 1667 as a treatment for mental illness, with predictably poor results. Its therapeutic utility and widespread use were initially limited by deficiencies in transfusion science and antisepsis. James Blundell, a British obstetrician, was recognized for the first allotransfusion in 1825. However, WBT did not become safe and therapeutic until the early 20th century, with the advent of reliable equipment, sterilization, and blood typing. The discovery of citrate preservation in World War I allowed a separation of donor from recipient and introduced the practice of blood banking. During World War II, Elliott and Strumia were the first to separate whole blood into blood component therapy (BCT), producing dried plasma as a resuscitative product for "traumatic shock." During the 1970s, infectious disease, blood fractionation, and financial opportunities further drove the change from WBT to BCT, with few supporting data. Following a period of high-volume crystalloid and BCT resuscitation well into the early 2000s, measures to avoid the resulting iatrogenic resuscitation injury were developed under the concept of damage control resuscitation. Modern transfusion strategies for hemorrhagic shock target balanced BCT to reapproximate whole blood. Contemporary research has expanded the role of WBT to therapy for the acute coagulopathy of trauma and the damaged endothelium. Many US trauma centers are now using WBT as a front-line treatment in tandem with BCT for patients suffering hemorrhagic shock. Looking ahead, it is likely that WBT will once again be the resuscitative fluid of choice for patients in hemorrhagic shock.

Controlled Manipulation and Active Sorting of Particles Inside Microfluidic Chips Using Bulk Acoustic Waves and Machine Learning.

Manipulation of cells, droplets, and particles via ultrasound within microfluidic chips is a rapidly growing field, with applications in cell and particle sorting, blood fractionation, droplet transport, and enrichment of rare or cancerous cells, among others. However, current methods with a single ultrasonic transducer offer limited control of the position of single particles. In this paper, we demonstrate closed-loop two-dimensional manipulation of particles inside closed-channel microfluidic chips, by controlling the frequency of a single ultrasound transducer, based on machine-vision-measured positions of the particles. For the control task, we propose using algorithms derived from the family of multi-armed bandit algorithms. We show that these algorithms can achieve controlled manipulation with no prior information on the acoustic field shapes. The method learns as it goes: there is no need to restart the experiment at any point. Starting with no knowledge of the field shapes, the algorithms can (eventually) move a particle from one position inside the chamber to another. This makes the method very robust to changes in chip and particle properties. We demonstrate that the method can be used to manipulate a single particle, three particles simultaneously, and also a single particle in the presence of a bubble in the chip. Finally, we demonstrate the practical applications of this method in active sorting of particles, by guiding each particle to exit the chip through one of three different outlets at will. Because the method requires no model or calibration, the work paves the way toward the acoustic manipulation of microparticles inside unstructured environments.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14days posttransplant. After 75min of kidney warm ischemia, four experimental groups of n=7 underwent 14h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240min of NMP with infusion of vehicle, 10million porcine, or 10million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

An automated centrifugal microfluidic assay for whole blood fractionation and isolation of multiple cell populations using an aqueous two-phase system.

Fractionating whole blood and separating its constituent components one from another is an essential step in many clinical applications. Currently blood sample handling and fractionation processes remain a predominantly manual task that require well-trained operators to produce reliable and reproducible results. Herein, we demonstrate an advanced on-chip whole human blood fractionation and cell isolation process combining (i) an aqueous two-phase system (ATPS) to create complex separation layers with (ii) a centrifugal microfluidic platform (PowerBlade) with active pneumatic pumping to control and automate the assay. We use a polyethylene glycol (PEG) and dextran (DEX) mixture as the two-phase density gradient media and our automated centrifugal microfluidic platform to fractionate blood samples. Different densities of precisely tuned PEG-DEX solutions were tested to match each of the cell types typically targeted during blood fractionation applications. By employing specially designed microfluidic devices, we demonstrate the automation of the following steps: loading of a whole blood sample on-chip, layering of the blood on the ATPS solution, blood fractionation, precise radial repositioning of the fractionated layers, and finally extraction of multiple, selected fractionated components. Fractionation of up to six distinct layers is shown: platelet-rich plasma, buffy coat, PEG, DEX with neutrophils, red blood cells (RBCs) and high density gradient media (HDGM). Furthermore, through controlled dispensing of HDGM to the fractionation chamber, we show that each of the fractionated layers can be repositioned radially, on-the-fly, without disturbing the interfaces, allowing precise transfer of target fractions and cell types into external vials via a chip-to-world interface. Cell counting analysis and cell viability studies showed equivalence to traditional, manual methods. An overall cell viability greater than 90% of extracted cells demonstrates that the proposed approach is suitable for cell isolation applications. This proof-of-principle demonstration highlights the utility of the proposed system for automated whole blood fractionation and isolation for blood cell applications. We anticipate that the proposed approach will be a useful tool for many clinical applications such as standard cell isolation procedures and other bioanalytical assays (e.g., circulating tumor cells, and cell and gene therapy).

Nucleic acid sample preparation from whole blood in a paper microfluidic device using isotachophoresis

Nucleic acid amplification tests (NAATs) are a crucial diagnostic and monitoring tool for infectious diseases. A key procedural step for NAATs is sample preparation: separating and purifying target nucleic acids from crude biological samples prior to nucleic acid amplification and detection. Traditionally, sample preparation has been performed with liquid- or solid-phase extraction, both of which require multiple trained user steps and significant laboratory equipment. The challenges associated with sample preparation have limited the dissemination of NAAT point-of-care diagnostics in low resource environments, including low- and middle-income countries. We report on a paper-based device for purification of nucleic acids from whole blood using isotachophoresis (ITP) for point-of-care NAATs. We show successful extraction and purification of target nucleic acids from large volumes (33 µL) of whole human blood samples with no moving parts and few user steps. Our device utilizes paper-based buffer reservoirs to fully contain the liquid ITP buffers and does not require complex filling procedures, instead relying on the natural wicking of integrated paper membranes. We perform on-device blood fractionation via filtration to remove leukocytes and erythrocytes from our sample, followed by integrated on-paper proteolytic digestion of endogenous plasma proteins to allow for successful isotachophoretic extraction. Paper-based isotachophoresis purifies and concentrates target nucleic acids that are added directly to recombinase polymerase amplification (RPA) reactions. We show consistent amplification of input copy concentrations of as low as 3 × 103 copies nucleic acid per mL input blood with extraction and purification taking only 30 min. By employing a paper architecture, we are able to incorporate these processes in a single, robust, low-cost design, enabling the direct processing of large volumes of blood, with the only intermediate user steps being the removal and addition of tape. Our device represents a step towards a simple, fully integrated sample preparation system for nucleic acid amplification tests at the point-of-care.

Transfusion de sang total : quelle place dans la réanimation du choc hémorragique traumatique ?

La transfusion sanguine s’est développée et a progressé au rythme des conflits militaires. La découverte des groupes sanguins et des règles de compatibilité croisée a permis d’identifier des « donneurs universels ». Le fractionnement du sang total et le stockage différencié de ses différents composants ont permis d’améliorer la sécurité transfusionnelle et la conservation des produits sanguins. &#x0D; Alors que la majorité des décès évitables en traumatologie sont liés à des hémorragies massives, les engagements militaires récents ont mis en évidence l’intérêt d’une réanimation transfusionnelle précoce et ciblée contre la coagulopathie. À la phase aiguë, globules rouges, facteurs de coagulation et plaquettes sont pour cette raison administrés selon une stratégie de ratios transfusionnels. Cette stratégie se heurte à des difficultés logistiques liées aux différentes conditions de conservation de ces produits, à leur délai de délivrance, et à la charge de travail que leur transfusion impose dans le contexte de l’urgence.&#x0D; Dans des environnements contraints, le prélèvement et la transfusion de sang total frais est resté une technique de recours qui a montré son intérêt clinique et logistique. Plusieurs équipes civiles et militaires ont démontré la possibilité de conserver à 4° C puis de transfuser du sang total de groupe O faiblement titré en hémolysine, afin d’obtenir une thérapeutique de réanimation transfusionnelle universelle et disponible sans délai.

Flow Rate and Low Hematocrit Measurements for In Vitro Blood Processing With Doppler Ultrasound.

In vitro techniques for the processing of flowing blood and its components have recently emerged from microfluidics. The blood flow rate and hematocrit are two keys parameters to monitor for guaranteeing the reliability of these techniques. But, there is a lack of monitoring methods adapted to low flow rates and small tubing. In this study, we exploit minimization approaches of continuous Doppler measurements to survey the blood flow rate. Combined with a packing factor model, we also estimate hematocrit from the Doppler spectrum. The presented method is implemented with a continuous-wave (CW) Doppler probe mounted on a 3D-printed support. The accuracy of the flow rate was measured in the range from 0.5 to 1.5 mL/min. For each of four different blood bags, hematocrit in the range under 8% was estimated from the Doppler spectrum using a packing factor model derived from the other three bags. Flow rate estimation shows a mean measurement error under 3% for a measurement time of 2 s. The mean error is still under 5% for a measurement time of 0.5 s. Hematocrit estimation for the four blood bags shows errors of 1.4%, 1.1%, 0.67%, and 0.70% Hct for a measurement time of 5 s. The versatility and simplicity of the method make it highly valuable for in vitro blood processing, in particular for low hematocrit blood fractionation techniques derived from microfluidics, as it can be performed through sterile tubing.

A method for the approximate determination of microparticle amount and mass in biological mixtures using a fractionation process in a centrifugal force field

The paper presents a simplified method of estimating the amount and specific mass of microparticles in complex biological mixtures using the fractionation process in the field of centrifugal forces. In the presented method, using the heaviest blood cells – erythrocytes – as an example, a geometric model of a binary fraction in a borderline equilibrium state formed in the process of blood fractionation was used. In this model, an approximated shape of the erythrocyte and a sample normal distribution of the size and number of microparticles in the examined model fraction filled with erythrocytes were used. Based on publicly available blood data, it has been shown that it is possible to estimate the amount and specific weight of microparticles. The accuracy of such estimation generally depends on the precision of representation of shapes, the degree of filling the fraction with microparticles and on the individual quantitative and dimensional distribution of these particles in the examined fraction. The above conclusions, which determine the accuracy of the method presented, were verified in the paper with the use of rheological blood data, commonly available in literature for various types of measuring containers, which affect the level of filling of a given fraction with microparticles. Keywords: fractionation of biological mixtures, fractionation of blood components, erythrocyte model, erythrocyte shape, blood components

Advances in biomarker detection: Alternative approaches for blood-based biomarker detection.

In the clinical setting, a blood sample is typically the starting point for biomarker search and discovery. Mass spectrometry (MS) is a highly sensitive and informative method for characterizing a very wide range of metabolites and proteins and is therefore a potentially powerful tool for biomarker discovery. However, the physicochemical characteristics of blood coupled with very large ranges of protein and metabolite concentrations present a significant technical obstacle for resolving and quantifying putative biomarkers by MS. Blood fractionation procedures are being developed to reduce the proteome/metabolome complexity and concentration ranges, allowing a greater diversity of analytes, including those at very low concentrations, to be quantified. In this chapter, several strategies for enriching and/or isolating specific blood components are summarized, including methods for the analysis of low and high molecular weight compounds, usually neglected in this type of assays, extracellular vesicles, and peripheral blood mononuclear cells (PBMCs). For each method, relevant practical information is presented for effective implementation.

Oscillatory inertial focusing in infinite microchannels

Inertial microfluidics (i.e., migration and focusing of particles in finite Reynolds number microchannel flows) is a passive, precise, and high-throughput method for microparticle manipulation and sorting. Therefore, it has been utilized in numerous biomedical applications including phenotypic cell screening, blood fractionation, and rare-cell isolation. Nonetheless, the applications of this technology have been limited to larger bioparticles such as blood cells, circulating tumor cells, and stem cells, because smaller particles require drastically longer channels for inertial focusing, which increases the pressure requirement and the footprint of the device to the extent that the system becomes unfeasible. Inertial manipulation of smaller bioparticles such as fungi, bacteria, viruses, and other pathogens or blood components such as platelets and exosomes is of significant interest. Here, we show that using oscillatory microfluidics, inertial focusing in practically "infinite channels" can be achieved, allowing for focusing of micron-scale (i.e. hundreds of nanometers) particles. This method enables manipulation of particles at extremely low particle Reynolds number (Rep < 0.005) flows that are otherwise unattainable by steady-flow inertial microfluidics (which has been limited to Rep > ∼10-1). Using this technique, we demonstrated that synthetic particles as small as 500 nm and a submicron bacterium, Staphylococcus aureus, can be inertially focused. Furthermore, we characterized the physics of inertial microfluidics in this newly enabled particle size and Rep range using a Peclet-like dimensionless number (α). We experimentally observed that α >> 1 is required to overcome diffusion and be able to inertially manipulate particles.