Washing Of Cells Research Articles

The Cultivation of Halophilic Microalgae Shapes the Structure of Their Prokaryotic Assemblages.

The influence of microalgae on the formation of associated prokaryotic assemblages in halophilic microbial communities is currently underestimated. The aim of this study was to characterize shifts in prokaryotic assemblages of halophilic microalgae upon their transition to laboratory cultivation. Monoalgal cultures belonging to the classes Chlorodendrophyceae, Bacillariophyceae, Trebouxiophyceae, and Chlorophyceae were isolated from habitats with intermediate salinity, about 100 g/L, nearby Elton Lake (Russia). Significant changes were revealed in the structure of algae-associated prokaryotic assemblages, indicating that microalgae supported sufficiently diverse and even communities of prokaryotes. Despite some similarities in their prokaryotic assemblages, taxon-specific complexes of dominant genera were identified for each microalga species. These complexes were most different among Alphaproteobacteria, likely due to their close association with microalgae. Other taxon-specific bacteria included members of phylum Verrucomicrobiota (Coraliomargarita in assemblages of Navicula sp.) and class Gammaproteobacteria (Salinispirillum in microbiomes of A. gracilis). After numerous washings of algal cells, only alphaproteobacteria Marivibrio remained in all assemblages of T. indica, likely due to a firm attachment to the microalgae cells. Our results may be useful for further efforts to develop technologies applied for industrial cultivation of halophilic microalgae and for developing approaches to obtain new prokaryotes with a microalgae-associated lifestyle.

P-103 Sperm selection for ICSI by rheotaxis and thermotaxis in In-Situ handmade microfluidics of fluidic walls: preliminary results of a Randomized Controlled Trial

Abstract Study question Does sperm selection by rheotaxis and thermotaxis in In-Situ handmade microfluidics of fluidic walls optimize ICSI outcomes and workflow? Summary answer In-Situ handmade rheo/thermotaxis microfluidics of fluidic walls is as effective and more time-efficient than centrifugation density gradients to support pre-implantation embryonic development to blastocyst. What is known already Microfluidics technologies stand as the latest sperm selection methodology for ICSI. To outline their application in ART we conceived a lab-on-a-chip approach for sperm selection by In-Situ handmade microfluidics of fluidic walls (isM) in the same ICSI plate which integrates two operative zones: one to allow seminal plasma dispersion and sperm thermotaxis and another one to allow sperm rheotaxis. Our microfluidics system can be prepared with any media suitable for ICSI, disregarding centrifugation, washing, plasticware and other cells. The isM protocol proved non-inferior to centrifugation density gradients (CDG) to support embryonic development and implantation in a previous sibling-oocyte study. Study design, size, duration The present RCT was approved by our ethical committee for research with medical products (CEIm approval number 2023/394). This preliminary analysis aims to detect deviations in the pre-defined primary outcomes: Normal Fertilization Rate, Day 5/6 usable blastocyst rate per MII, and per zygote. This analysis includes data from 21 couples undergoing an ICSI treatment in our center during December 2023-January 2024. Only patients without clinical or analytical suspicion of sperm DNA fragmentation were included. Participants/materials, setting, methods Patients were randomly allocated to isM (n 9) or to CGD (n 12) groups. All participants signed an informed consent. Sperm samples were processed directly after liquefaction. For CGD, samples were prepared in 80-40% PureSperm® layers by isopycnic centrifugation followed by washing and dilution steps. The isM system was prepared by displaying three microdroplets of different volumes following a L-shape interconnection and allowing a temperature gradient of 2ºC between the first and the second microdroplet. Main results and the role of chance Sperm characteristics showed the following total mean values for the isM and CGD groups, respectively: concentration (58,2±38,0 × 106/ml and 52,7±29,1 × 106/ml) and progressive sperm concentration (16,6±17,0 × 106/ml and 16,8±13,4 × 106/ml). Women average age was 36,7±2,9 isM and 35,8±2,5 CDG. This analysis includes a total of 168 MII oocytes (n = 69 isM and n = 99 CDG), with a mean number of MII oocytes per patient of 7,4±2,1 isM and 8,3±1,9 CDG. Comparisons between the two groups showed that isM protocol optimizes ICSI outcomes by means of Normal Fertilization Rate: 88,4% vs 76,8%; p < 0,05, Day 5 A/B grade blastocyst rate: 36,1 vs 31,6%; ns, Day 5/6 usable blastocyst rate per MII: 58,0% vs 44,4%; ns and Day 5/6 usable blastocyst rate per diploid zygote: 65,6% vs 57,9%; ns. The isM protocol also optimizes the IVF workflow compared to CDG, as the time to obtain sperm for ICSI after liquefaction was 26 vs 77 minutes, respectively. Limitations, reasons for caution The study aims for a total size of 50 ICSI cycles to validate statistically our data at a power of 80%. The isM protocol depends on a free hand preparation. A ready-to-use surface would benefit the operability, albeit the use of templates and the training reduces inter and intra-operator variability. Wider implications of the findings The potential benefits of the isM protocol on fertilization could be significant for patients with a limited number of available oocytes for ICSI. According to the literature, microfluidics reduce the DNA fragmentation index in the selected sperm fraction. The isM method could be especially beneficial in patients with this pathology. Trial registration number NCT number assignment pending

Micro-flow cell washing technique combined with single-cell Raman spectroscopy for rapid and automatic antimicrobial susceptibility test of pathogen in urine

Facing the rapid spread of antimicrobial resistance, methods based on single-cell Raman spectroscopy have proven their advances in reducing the turn-around time (TAT) of antimicrobial susceptibility tests (AST). However, the Raman-based methods are still hindered by the prolonged centrifugal cell washing procedure, which may require complex labor operation and induce high mechanical stress, resulting in a pretreatment time of over 1 h as well as a high cell-loss probability. In this study, we developed a micro-flow cell washing device and corresponding Raman-compatible washing chips, which were able to automatically remove the impurities in the samples, retain the bacterial cell and perform Raman spectra acquisition in situ. Results of washing the 5- and 10-μm polymethyl methacrylate (PMMA) microspheres showed that the novel technique achieved a successful removal of 99 % impurity and an 80 % particle retention rate after 6 to 10 cycles of washing. The micro-flow cell washing technique could complete the pretreatment for urine samples in a 96-well plate within 10 min, only taking 15 % of the handling time required by centrifugation. The AST profiles of urine sample spiked with E. coli 25922, E. faecalis 29212, and S. aureus 29213 obtained by the proposed Raman-based approach were found to be 100 % consistent with the results from broth micro-dilution while reducing the TAT to 3 h from several days which is required by the latter. Our study has demonstrated the micro-flow cell washing technique is a reliable, fast and compatible approach to replace centrifuge washing for sample pretreatment of Raman-AST and could be readily applied in clinical scenarios.

Abstract 6331: A novel automated platform for rapid manufacturing of CAR T cells

Abstract Autologous cell therapy using genetically modified T cells to express chimeric antigen receptor (CAR) has yielded durable responses in cancers patients. However, currently only a small portion of patients are privileged to receive the treatment. High cost and long manufacturing time contribute to the key limitations prohibiting cell therapy broad adoption. Researchers discovered that reducing ex vivo culture improves anti-tumor activity of CAR T cells1 and recently demonstrated the process of generating functional CAR T cells within 24 hours2. This rapid process provides great promise in cost reduction of CAR T therapy and opportunity to have cells manufactured in close-to-patient settings. CART cell manufacturing process involves: (1) collection of peripheral blood; (2) isolation of T cells; (3) performing transduction via viral vector; (4) washing CAR T cells to remove viral vector, and (5) harvesting CAR T cells in IV fluid. Conventional process uses magnetic separation and serial centrifugation technologies on multiple platforms with manual operations. Here we demonstrate the feasibility of rapid manufacturing CAR T cells on an automated platform with closed fluidics without centrifugation steps. The platform, MARS® Atlas, is built with innovative technologies in immune-magnetic cell isolation and acoustic cell washing and implemented as a modular system. In this feasibility test, T cells were isolated from peripheral blood from a healthy donoron MARS® magnetic module. eGFP expressing lentivirus were added to the enriched T cells. After short incubation, cells and virus were washed with fresh medium on the MARS® acoustic module and cells were incubated with virus overnight without activation. Infected cells were washed on the acoustic module to remove free virus. Product CAR T cells were harvested in saline solution and ready for QC analysis. Starting from 20mL of peripheral blood, magnetic T cell separation was done 2mL/min achieving 90% T cell purity, 90% recovery. In the first acoustic wash, medium exchange of the mixture of cells and virus took place at 0.9mL/min flow rate. As a result, 90% removal of contaminating molecule and 99.6% recovery of T cells were achieved. The second acoustic wash completed medium exchange of transfected cells at 0.9mL/min with 96.7% removal of virus and 90% CAR T cell recovery. As a result, 18 million GFP expressing T cells were produced. In this study we have demonstrated the feasibility of rapidly manufacturing CAR-T cells on an automated platform MARS® Atlas. This result indicates the MARS® Atlas being a potential CAR T manufacturing platform to be used in close-to-patient settings to enable low-cost cell therapy. 1 Saba Ghassemi, Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells, Cancer Immunol Res; 6(9) September 2018 2 Saba Ghassemi, Rapid manufacturing of non-activated potent CAR T cells, Nature Biomedical Engineering | VOL 6 | February 2022 | 118-128 Citation Format: Silin Sa, Ethan Nguyen, Jie Pan, Liping Yu. A novel automated platform for rapid manufacturing of CAR T cells [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 6331.

DNA framework signal amplification platform-based high-throughput systemic immune monitoring

Systemic immune monitoring is a crucial clinical tool for disease early diagnosis, prognosis and treatment planning by quantitative analysis of immune cells. However, conventional immune monitoring using flow cytometry faces huge challenges in large-scale sample testing, especially in mass health screenings, because of time-consuming, technical-sensitive and high-cost features. However, the lack of high-performance detection platforms hinders the development of high-throughput immune monitoring technology. To address this bottleneck, we constructed a generally applicable DNA framework signal amplification platform (DSAP) based on post-systematic evolution of ligands by exponential enrichment and DNA tetrahedral framework-structured probe design to achieve high-sensitive detection for diverse immune cells, including CD4+, CD8+ T-lymphocytes, and monocytes (down to 1/100 μl). Based on this advanced detection platform, we present a novel high-throughput immune-cell phenotyping system, DSAP, achieving 30-min one-step immune-cell phenotyping without cell washing and subset analysis and showing comparable accuracy with flow cytometry while significantly reducing detection time and cost. As a proof-of-concept, DSAP demonstrates excellent diagnostic accuracy in immunodeficiency staging for 107 HIV patients (AUC > 0.97) within 30 min, which can be applied in HIV infection monitoring and screening. Therefore, we initially introduced promising DSAP to achieve high-throughput immune monitoring and open robust routes for point-of-care device development.

The Bioluminescent Modified Ham Test Identifies the Classical Pathway As the Major Driver of Complement Activation in Atypical Hemolytic Uremic Syndrome and Primary Antiphospholipid Syndrome

Introduction: The role of complement is increasingly recognized in disease pathogenesis, however diagnostic assays are limited by low predictive value, technical requirements, and inability to assess relative contributions of each pathway of complement (classical, alternative, or lectin). The traditional mHam was developed as a functional assay of complement-mediated eukaryotic cell death in atypical hemolytic uremic syndrome (aHUS). However, broader use of the assay has been limited by inter-operator dependence and technical requirements. We have developed a bioluminescent mHam that is robust and easily performed (no cell washes or exogenous reagent addition) and utilizes an autonomously bioluminescent HEK293 cell line to allow for real-time assessment of changes in metabolic health due to complement activation. Given both HEK293 and endothelial cells lack complement receptor 1 (CR1), a crucial regulator of the classical pathway (CP), this assay measures complement activity of greater relevance to disease pathogenesis. Methods: The bioluminescent mHam uses LiveLight (LL) HEK293 cells (490 BioTech) engineered to stably express the bacterial luciferase ( lux) and flavin reductase genes ( luxF) to create autonomous bioluminescence. A PIGA null clone (LL PIGA- HEK293) was created through proaerolysin selection (removing GPI-anchored complement regulators CD55 and CD59). To perform the assay, 40,000 cells were harvested with trypsin/EDTA and plated as triplicates in gelatin veronal buffer (GVB ++,80 µL). Serum (20 µL) was added after specified treatments (heat inactivation or inhibitor addition). Activity was monitored by serial luminescence measurements every 5 min at 37 °C. A relative luminescence of <25% at one hour is designated as positive based upon lowest replicate of any healthy control. Results: The bioluminescent mHam, unlike the original mHam, provides continuous measurement of complement activity (example tracing Figure 1). The complement specificity of the luminescent changes were demonstrated with heat inactivation, terminal complement blockade with eculizumab, and calcium/magnesium dependence. The relative contribution of CP to changes in luminescence was confirmed with the specific CP inhibitor sutimlimab as well as use of pathway specific buffers. Healthy control (HC) samples show a relative luminescence of 53.7% (95%CI 44.0-63.4) with no difference observed in acute thrombotic thrombocytopenic purpura (TTP) (mean 56.2%, 95%CI 41.2-71.2, p=>0.9999). We next utilized samples of aHUS patients known to be in remission, off complement inhibitor therapy. A large proportion of these patients have ongoing assay positivity even in the traditional mHam (9 of 13 in one unpublished cohort). The bioluminescent mHam shows that 5 of 6 aHUS patients in remission have ongoing positivity (mean 17.56%, 95%CI -10.5-45.6, p=0.0131). An acute aHUS sample showed similar results (17.8% after plasmapheresis). Unexpectedly, the relative luminescence is rescued by addition of either C1s or C5 inhibitors, but use of alternative pathway (AP) inhibitors has no significant rescuing effect ( Figure 1-2). Of aHUS patients tested while on complement inhibition, 91% (10/11) show greater than 88% blockade ( Figure 2 aHUS on therapy) . Finally, in one cohort, the traditional mHam was positive in 52% (14/27) of primary antiphospholipid syndrome (PAPS) cases and 15% (10/66) of secondary APS (SAPS) (unpublished data). Thus far, the bioluminescent mHam shows abnormal complement activity in 92% (12/13) of PAPS and 17% (2/12) of patients in SAPS ( Figure 2). Again, complement inhibition with C1s or C5 inhibitors abrogated the effect in a dose dependent manner. Conclusions: The bioluminescent mHam test is an exquisitely sensitive real-time measure of abnormal complement activity. It is easy to perform, more closely approximates the complement regulators on renal endothelium, can be utilized for therapeutic monitoring, and has already offered novel disease insights through the use of specific pathway inhibitors. Although aHUS is traditionally categorized as a disorder of the AP, these results suggest the “trigger” mediating disease pathology is the classical pathway. Also, we show that a large proportion of primary APS patients have abnormal CP activation responsive to C1s and C5 inhibition, suggesting the disease may be responsive to complement-based therapeutics.

Feasibility of Extracorporeal Delivery of Fusosomes to Generate CAR T Cells In Vivo

Fusosomes are viral vectors pseudotyped with modified paramyxovirus envelopes targeting specific cell types. A CD8-targeted fusosome delivering a CD19CAR transgene has the potential to provide an off-the-shelf therapeutic approach to generate in vivo CD19-directed chimeric antigen receptor (CAR) T cells in patients. For an in vivo gene delivery platform there are several approaches for administration that could be considered including direct intravenous (IV) administration or extracorporeal delivery (ECD). With ECD, patients would first undergo apheresis, then fusosomes would be retained with patient peripheral blood mononuclear cells (PBMCs) in the apheresis bag for a brief period of time before the entire contents of the bag is IV delivered into the same patient. Here, we demonstrate the feasibility of this approach to generate in vivo CAR T cells in a clinical setting with a mock infusion and discuss the translation of these findings to direct IV administration. A healthy donor was apheresed to collect PBMCs using standard apheresis equipment. No further cell enrichment, separation, or washing was performed. CD8-targeted fusosome formulated in an isotonic cryoprotective buffer was administered into the apheresis bag by gravity via an infusion set at a dose of ~0.5 IU/cell, gently mixed and retained for 30 min. The contents of the bag were mock infused into sample collection bags at a controlled flow rate using an infusion pump. Samples were collected before and after fusosome addition, after 30 min retention, and every 30 min during the mock infusion. Cell counts and viability were measured by complete blood count (CBC) and flow cytometry analyses. Fusosome binding to CD8 + T cells was measured by flow cytometry. Cytokines and complement components in supernatants were measured by ELISA or MSD. Residual fusosome in the supernatant was measured by ddPCR. Transduction of CD8 + T cells was measured by flow cytometry and vector copy number (VCN) analysis after PBMCs were washed, activated with CD3/CD28 beads, and cultured for 8 days. Early transcription events were measured by ddPCR using specific amplicons targeting different DNA species generated during reverse transcription. The CD8-targeted fusosome was successfully deliveredusing aseptic techniques to a freshly collected apheresis bag.PBMC viability stayed high (>%95) throughout the process. There were no major changes in white blood cell, platelet, or red blood cell levels as measured by CBC analysis. There were no changes in CD8 + T cell phenotype throughout the time course. Fusosome binding to CD8 + T cells was detected as early as fusosome addition to the apheresis bag (~5 min) and increased over the course of the process. Analysis of vector genomes in the supernatants indicated presence of unbound residual fusosome. Exposure of PBMC to fusosome did not induce cytokine secretion or complement activation throughout the process. Early events of transduction of cells could be detected by analysis of reverse transcription intermediates. Analysis of cultured cells via both flow cytometry and VCN analysis indicated generation of CD8 + CAR T cells upon short-term exposure to fusosome. In vivo delivery ofCD8-targeted fusosome encoding a CD19CAR transgene enables a novel CAR T cell approach. This study demonstrated that administration of fusosomes via ECD is operationally feasible in a clinical setting and scale as a novel method of administration to generate CD8 + CD19CAR T cells in vivo. ECD setup also served as an in vitro pharmacokinetic model for direct IV infusion. In scenarios where in vivo exposure is limited due to very rapid clearance (e.g. few minutes), ECD has the benefit of reducing the volume of distribution and bringing T cells and fusosomes in close proximity for efficient gene delivery. The findings of study suggest that with rapid binding of fusosomes to CD8 + T cell leading to early transcription events, and an in vivo half-life of more than 30 min, fusosome may generate CAR T cells successfully via direct IV administration.

FRI540 A Novel, Rapid, Sensitive Bioassay For Thyrotropin Receptor Stimulatory Antibodies Improves Diagnosis & Management Of Autoimmune Thyroid Disease

Abstract Disclosure: G.J. Kahaly: None. M.A. Lupo: None. A.T. Bossowski: None. L. Frommer: None. B. Hatun: None. A. George: None. J. Wolf: None. Background: Autoantibody mimicry of hormone action at the thyrotropin receptor (TSH-R) and aberrant signaling of TSH-R by autoantibodies (TSH-R-Ab) causes autoimmune thyroid disease (AITD), hyperthyroidism and hypothyroidism, both of which affect millions of patients worldwide. In this multicenter, single blind study, the specificity, sensitivity and performance of a novel bioassay for stimulatory TSH-R-Ab were tested. Methods: TSH-R-Ab were measured in a blinded manner with a TSH-R binding immunoassay (Cobas e411, Roche), and both stimulatory (TSAb) and blocking (TBAb) bioassays with luciferase release as readout (Thyretain®, Quidel) according to manufacturer’s instructions. TSH-R-Ab was also measured using the new TSAb bioassay (TurboTM). TurboTM utilizes a cAMP biosensor that is an engineered form of firefly luciferase in which the cAMP-binding domain of protein kinase A is fused between the N- and C-termini such that the luciferase is inactive until cAMP binds to the cAMP binding site. Using this biosensor, luciferase activity is proportional to intracellular cAMP levels. TurboTM detects TSAb in serum at room temperature, in a real-time homogeneous format, which delivers results in 60 minutes. It does not require cell culture, sample dilution, washing or cell lysis steps. Results: Eight-hundred forty-four samples of unselected, consecutive AITD patients and control subjects, devoid of autoimmune endocrine and thyroid disorders, were included. Mean age (SD) was 49 (14.7) years, range 1-89 years. Female: male sex ratio was 3.7:1. The four TSH-R-Ab assays were negative in all 174 controls. In contrast, the Turbo TSAb, Thyretain® TSAb and the binding assays detected TSAb in 557 of 670 (83%), 526/670 (78%) and 430/637 (67%) AITD samples, respectively (TurboTM bioassay versus binding immunoassay, chi-square test p<0.001). The results of the Turbo bioassay highly correlated with both thyroid function in Graves’ hyperthyroidism as well as with the clinical activity and severity of associated thyroid eye disease (p<0.001). Inter- (two users) and intra- (two lots) assay comparison for the Turbo bioassay showed coefficient of variations of only 4%, 3%, and 2%, respectively. The two TSAb bioassays, TurboTM and Thyretain®, correlated (Spearman’s r =0.7, p<0.001). Furthermore, the magnitude of serum levels in both TSAb bioassays correlated with TSH-R-Ab binding (both p<0.001). Finally, seventeen AITD samples showed dual TSH-R-Ab positivity in the TBAb and TSAb bioassays. Conclusions: The novel, rapid, easy-to-perform, sensitive and specific TurboTM bioassay performs better than the established TSH-R-Ab binding immunoassay and the Thyretain® bioassay. It provides clinically useful information for the accurate diagnosis and management of AITD patients. Presentation: Friday, June 16, 2023

A review of acoustofluidic separation of bioparticles.

Acoustofluidics is an emerging interdisciplinary research field that involves the integration of acoustics and microfluidics to address challenges in various scientific areas. This technology has proven to be a powerful tool for separating biological targets from complex fluids due to its label-free, biocompatible, and contact-free nature. Considering a careful designing process and tuning the acoustic field particles can be separated with high yield. Recently the advancement of acoustofluidics led to the development of point-of-care devices for separations of micro particles which address many of the limitations of conventional separation tools. This review article discusses the working principles and different approaches of acoustofluidic separation and provides a synopsis of its traditional and emerging applications, including the theory and mechanism of acoustofluidic separation, blood component separation, cell washing, fluorescence-activated cell sorting, circulating tumor cell isolation, and exosome isolation. The technology offers great potential for solving clinical problems and advancing scientific research.

Continuous On-Chip Cell Washing Using Viscoelastic Microfluidics.

Medium exchange of particles/cells to a clean buffer with a low background is essential for biological, chemical, and clinical research, which has been conventionally conducted using centrifugation. However, owing to critical limitations, such as possible cell loss and physical stimulation of cells, microfluidic techniques have been adopted for medium exchange. This study demonstrates a continuous on-chip washing process in a co-flow system using viscoelastic and Newtonian fluids. The co-flow system was constructed by adding a small amount of biocompatible polymer (xanthan gum, XG) to a sample containing particles or cells and introducing Newtonian fluids as sheath flows. Polymer concentration-dependent and particle size-dependent lateral migration of particles in the co-flow system were examined, and then the optimal concentration and the critical particle size for medium exchange were determined at the fixed total flow rate of 100 μL/min. For clinical applications, the continuous on-chip washing of white blood cells (WBCs) in lysed blood samples was demonstrated, and the washing performance was evaluated using a scanning spectrophotometer.

Long-Read Nanopore Sequencing of RPGR ORF15 is Enhanced Following DNase I Treatment of MinION Flow Cells.

RPGR ORF15 is an exon present almost exclusively in the retinal transcript of RPGR. It is purine-rich, repetitive and notoriously hard to sequence, but is a hotspot for mutations causing X-linked retinitis pigmentosa. Long-read nanopore sequencing on MinION and Flongle flow cells was used to sequence RPGR ORF15 in genomic DNA from patients with inherited retinal dystrophy. A flow cell wash kit was used on a MinION flow cell to increase yield. Findings were confirmed by PacBio SMRT long-read sequencing. We showed that long-read nanopore sequencing successfully reads through a 2 kb PCR-amplified fragment containing ORF15. We generated reads of sufficient quality and cumulative read-depth to detect pathogenic RP-causing variants. However, we observed that this G-rich, repetitive DNA segment rapidly blocks the available pores, resulting in sequence yields less than 5% of the expected output. This limited the extent to which samples could be pooled, increasing cost. We tested the utility of a MinION wash kit containing DNase I to digest DNA fragments remaining on the flow cell, regenerating the pores. Use of the DNase I treatment allowed repeated re-loading, increasing the sequence reads obtained. Our customised workflow was used to screen pooled amplification products from previously unsolved inherited retinal disease (IRD) in patients, identifying two new cases with pathogenic ORF15 variants. We report the novel finding that long-read nanopore sequencing can read through RPGR-ORF15, a DNA sequence not captured by short-read next-generation sequencing (NGS), but with a more reduced yield. Use of a flow cell wash kit containing DNase I unblocks the pores, allowing reloading of further library aliquots over a 72-h period, increasing yield. The workflow we describe provides a novel solution to the need for a rapid, robust, scalable, cost-effective ORF15 screening protocol.

Feasibility of an acoustophoresis-based system for a high-throughput cell washing: application to bioproduction

Background aimsThese last decades have seen the emergence and development of cell-based therapies, notably those based on mesenchymal stromal cells (MSCs). The advancement of these promising treatments requires increasing the throughput of processed cell for industrialization in order to reduce production costs. Among the various bioproduction challenges, downstream processing, including medium exchange, cell washing, cell harvesting and volume reduction, remains a critical step for which improvements are needed. Typically, these processes are performed by centrifugation. However, this approach limits the automation, especially in small batch productions where it is performed manually in open system. MethodsAn acoustophoresis-based system was developed for cell washing. The cells were transferred from one stream to another via the acoustic forces and were collected in a different medium. The optimal flow rates of the different streams were assessed using red blood cells suspended in an albumin solution. Finally, the impact of acoustic washing on adipose tissue-derived MSCs (AD-MSCs) transcriptome was investigated by RNA-sequencing. ResultsWith a single passage through the acoustic device at input flow rate of 45 mL/h, the albumin removal was up to 90% while recovering 99% of RBCs. To further increase the protein removal, a loop washing in two steps was performed and has allowed an albumin removal ≥99% and a red blood cell/AD-MSCs recovery of 99%. After loop washing of AD-MSCs, only two genes, HES4 and MIR-3648-1, were differently expressed compared with the input. ConclusionsIn this study, we developed a continuous cell-washing system based on acoustophoresis. The process allows a theoretically high cell throughput while inducing little gene expression changes. These results indicate that cell washing based on acoustophoresis is a relevant and promising solution for numerous applications in cell manufacturing.

Separation and Washing of Candida Cells from White Blood Cells Using Viscoelastic Microfluidics

An early and accurate diagnosis of Candida albicans is critical for the rapid antifungal treatment of candidemia, a mortal bloodstream infection. This study demonstrates viscoelastic microfluidic techniques for continuous separation, concentration, and subsequent washing of Candida cells in the blood. The total sample preparation system contains two-step microfluidic devices: a closed-loop separation and concentration device and a co-flow cell-washing device. To determine the flow conditions of the closed-loop device, such as the flow rate factor, a mixture of 4 and 13 μm particles was used. Candida cells were successfully separated from the white blood cells (WBCs) and concentrated by 74.6-fold in the sample reservoir of the closed-loop system at 800 μL/min with a flow rate factor of 3.3. In addition, the collected Candida cells were washed with washing buffer (deionized water) in the microchannels with an aspect ratio of 2 at a total flow rate of 100 μL/min. Finally, Candida cells at extremely low concentrations (Ct > 35) became detectable after the removal of WBCs, the additional buffer solution in the closed-loop system (Ct = 30.3 ± 1.3), and further removal of blood lysate and washing (Ct = 23.3 ± 1.6).

A comparative study between gel card method and manual method for Coomb\'s test

The main objective of this study is to compare the Gel Card method and the Conventional Tube method for Coomb’s test. The standard procedures were being followed while performing the above mentioned two test. Based on an 8 months study, Gel card method was proven to be more reliable in concordance with its calculated p-value and the sensitivity. The advantages and disadvantages of which have been mentioned in the following. For Gel Card method, the principle of saphadex gel as a semi-solid medium is being used to trap any agglutination. For the Conventional Tube method, the SOP was being followed involving cell washing and confirmation via microscopy for any micro-clumps. The use of polyspecific antiglobulin was implemented for both the above test. The study showed p-value for Gel Card method to be < 0.05 which proves to be significant and the sensitivity of Gel Card method was also better compared to the Conventional Tube method. Our study showed that gel card is more sensitive, easy to perform and lesser time consumption, lesser sample volume plus standardized reporting. Results of Gel card can be preserved for 3-4 days. Avoidance of interobserver variability is also an added benefit due to the standard grading system. It is therefore an excellent method for detecting agglutination compared to the Conventional Tube method.

Selective immunoglobulin A deficiency in a sample of Iraqi blood donors in the National Blood Transfusion Center-Baghdad

BACKGROUND: Selective immunoglobulin (Ig) A deficiency is the most common Ig deficiency in the world. Serum IgA level is reduced, 7 mg/dL (70 μg/mL), thus, associating with a wide range of clinical symptoms and signs that are related directly and indirectly to disturbances in the body immune system. Selective IgA deficiency patients are known to develop antibodies against lacking IgA. Whenever these antibodies are found in an individual's circulating blood, a severe allergic response and anaphylaxis can ensue upon receiving IgA containing blood or blood components. Hence, certain protocols are recommended in populations with increased prevalence of selective IgA deficiency including the use of red blood cell (RBC) washing machines or IgA-deficient blood components. Both measures are costly and require special maintenance procedures.objectives: The aim of this study was to screen for the presence of selective IgA deficiency in a group of blood donors and its probable role in the events of transfusion reactions that frequently occur which may necessitate the application of protocols that involve sophisticated procedures and highly-demanding machines.SUBJECTS AND METHODS: This was a laboratory study that included 300 volunteer blood donors in the National Blood Transfusion Center. They were screened for their serum IgA levels. Related questionnaire was conducted and chemiluminescence immunoassay technique with MAGLUMI 600 series machine was used. This machine is already used by the National Center of Teaching Laboratories for variety of biochemical and immunological assays. Serum IgA levels then were plotted against age, blood group and smoking status.RESULTS: No single case of Selective IgA deficiency was documented. Serum IgA levels showed increased levels with age and smoking demonstrated no significant effect on the Ig levels.CONCLUSION: In light of these results, it does not seem urgent or necessary at this point of time to introduce the highly demanding and expensive RBC washing machines in blood banks in Iraq. We recommend conducting a larger study that is more comprehensive with wider involvement of population, considering age, gender, and ethnicity.

Perbedaan Hasil Derajat Aglutinasi Serum Grouping Tube Test Dengan Suspensi Reagen NaCl 0,9% Siap Pakai dan Suspensi Reagen NaCl 0,9% Dari Garam Dapur

ABO blood group examination is a simple laboratory test performed to determine a person’s blood group. There are two types of ABO blood group test, namely cell grouping and serum grouping. Serum grouping examination is very important to prevent incompatible transfusion reactions and organ transplant rejection. Serum grouping was carried out using 10% saline wash suspension of cell A, B and O. The blood cell washing stage was used to remove fragile cells and interfering substances that could result in errors in the examination. The main ingredient used in the blood cell washing procedur is 0.9% NaCl. This study aims to determine the degree of agglutination serum grouping tube test with ready to use 0.9% NaCl reagent suspension and 0.9% NaCl reagent suspension from table salt. The type of research is an experiment. The study was conducted at UDD PMI South Sumatra Province on 30 December 2021 to 5 January 2022 with a totalsample of 67 samples. The results showed that the average degree of agglutination the serum grouping tube test with 0.9% NaCl reagent ready to use was 3.73 and the average degree of agglutination the serum grouping tube test with0.9% NaCl reagent from table salt was 3.69. Wilcoxon test results obtained p value = 0.450. The conclusion of the study, that there is no differences in the results of the agglutination serum grouping tube test with ready to use 0.9% NaCl reagen suspension and 0.9% NaCl reagent suspension from table salt. The table salt showed that 0.9% NaCl reagent can be used as alternative reagent NaCl 0.9% ready to use in the examination of Iserum grouping tube test.

(Digital Presentation) Design, Flow Simulation and Experimental Test of Micro/Milibioreactor with Pillar Structures for Bubble Control and Cell Growth

The demand for Lab on a Chip (LOC) devices, such as microbioreactors, has driven microfluidic research and technology. With the capability of controlling the environment and monitoring results in real-time, microfluidic devices are highly advantageous in the application of cell culture. On the other hand, micro/nano-scale bubbles have become a field of growing interest for addressing many challenges in LOC applications. In this work, we present a novel microfluidic bioreactor based on a micropillars design that reduces stresses caused by bubbles on the cell growth in the microchip. The combination of one chamber with 3-pillars and 10-pillars design with the channel heights of 270 µm and 2 mm was evaluated.To obtain the optimal architecture, computational fluid dynamics (CFD) simulations using SolidWorks Flow Simulation Software were created to characterize the designs. Simulations are essential to assure that the washing of cells and bubbles do not occur in the fluid and to weeding out the designs that are suitable for cell growth. Numerical results showed that the velocity is high at the inlet and outlet when compared to the interior portions of the chamber. A higher flow rate and velocity were observed during the filling time in the 10-pillars design (3.0 x10-4 m/sec) in comparison with the 3-pillars design (2.4 x 10-4 m/sec). Also, a more homogeneous filling pattern was observed in the 3-pillars designs. This behavior occurs because the increased number of pillars manifests with a greater number of nuclei of flow disturbance, and more centers of agitation, resulting in a more heterogeneous flow. Pressure is homogenous for both 3 and 10 Pillars designs with slight changes from the inlet, with higher pressure, to the outlet, with lower pressure.The microfluidic devices were manufactured using flexographic master mold (Fmold) technology and polydimethylsiloxane (PDMS 10:1) as substrate. The total liquid capacity of the microchips was calculated as 205.5 μL for 270-um-height and 1522 μL for 2-mm-height. An experimental flow test with methylene blue using the syringe pump, with an inlet flow rate at 2.5 μL/sec, and image analysis from video using ImageJ software was performed. The experimental results were found to be comparable to the simulations results. When the number of pillars and height of the chambers increased, a higher flow rate and velocity were observed during the filling time. In this context, the 3-pillar design with 2 mm height was considered the most suitable design for cell culture applications. In addition, the 2 mm larger height design was tested for bubble control, and the results showed some of the bubbles were easily removed by applying flow rate, and the ones that remained inside the chamber did not block the channels in the pillar’s designs without affecting the system.The present platform has a simple architecture, inexpensive manufacturing methods, ease of operation, and the ability to use different types of cells. The pillar milli-bioreactor system has the advantages of bubble control, a large volume of work, real-time monitoring, and a suitable microenvironment for cell culture with prospects for future health applications.

Cell Tracking by Magnetic Particle Imaging: Methodology for Labeling THP-1 Monocytes with Magnetic Nanoparticles for Cellular Imaging.

Magnetic particle imaging (MPI) is a noninvasive tomographic imaging modality for the quantitative visualization of magnetic nanoparticles (MNPs) with high temporal and spatial resolution. The general capability of MPI for cell tracking (e.g., monitoring living cells labeled with MNPs) has successfully been shown. MNPs in cell culture media are often subjected to structural and magnetic changes. In addition to the deteriorating reproducibility, this also complicates the systematic study of the relationship between the MNP properties and their cellular uptake for MPI. Here, we present a method for the preparation of magnetically labeled THP-1 (Tamm–Horsfall Protein-1) monocytes that are used in MPI cell tracking. The method development was performed using two different MPI tracers, which exhibited electrostatic and steric stabilizations, respectively. In the first step, the interaction between the MNPs and cell culture media was investigated and adjusted to ensure high structural and magnetic stability. Furthermore, the influences of the incubation time, MNP concentration used for cellular uptake, and individual preparation steps (e.g., the washing of cells) were systematically investigated. Finally, the success of the developed loading method was demonstrated by the MPI measurements. The presented systematic investigation of the factors that influence the MNP loading of cells will help to develop a reliable and reproducible method for MPI monocyte tracking for the early detection of inflammation in the future.

Towards single cell ICP-MS normalized quantitative experiments using certified selenized yeast

In the search for a normalized procedure to replicate and compare single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) experiments, SELM-1, a certified reference material containing selenium enriched yeast cells has been used. Selenium concentrations (both, intra- and extracellular) have been measured using either sequential or simultaneous procedures. Regarding quantitative results, the sequential procedure involving cell washing followed by freeze drying of the washed material and intracellular Se quantification using SC-ICP-MS provided best results. In this case, intracellular Se accounted for 1304 ± 48 mg kg−1 (corresponding to 64% of the certified Se content). The average mass of Se per yeast cell was 41.6 fg Se with a dispersion of 1.6–279 fg Se/cell. In the isolated extracellular Se fraction, the Se concentration accounted for 412 ± 48 mg kg−1 (about 21% of the total Se). Thus, the sequential procedure provided a total Se recovery of about 85% with respect to the certified value. The direct dilution and simultaneous measurement of intra- and extracellular Se by SC-ICP-MS provided results of 1024 ± 42 mg kg−1 for intracellular and 316 ± 30 mg kg−1 for extracellular Se representing a total recovery of about 66%. In both cases, an initial thorough characterization of the cell density per solid weighed material was conducted by flow cytometry and the cell integrity ensured using confocal microscopy. These results clearly demonstrated that with appropriate sample preparation, SC-ICP-MS is a unique tool, which is capable of providing quantitative information about intracellular and extracellular Se. In addition, SELM-1 seems the ideal tool to enable data normalization at the single cell level to replicate, benchmark, and improve new SC-ICP-MS studies by using the same material for data validation.

Evaluation of the Post-Antifungal Effect of Rezafungin and Micafungin against Candida albicans, Candida parapsilosis and Candida glabrata.

Rezafungin, a new echinocandin with an extended half-life, exhibits potent activity against Candida spp. Aside from the MIC, specific interactions between antifungal and isolate, including the duration of anti-infective activity, may impact dose interval choices and infection outcome. We evaluated rezafungin and micafungin post-antifungal effect (PAFE) against C.albicans, C.parapsilosis and C.glabrata. Six Candida spp. isolates were tested, including two of each species, C.albicans, C.parapsilosis and C.glabrata. Antifungal susceptibility testing was performed using the CLSI reference broth microdilution method. Antifungal concentrations of 1x, 4x and 16x the baseline MIC were used for PAFE determinations. Colony counts were performed at T0 (pre-exposure), after the 1-h drug exposure, after the cell wash (T1), and at T2, T4, T8, T12, T24 and T48h. Rezafungin PAFE results were equivalent to micafungin PAFE values for one C.albicans (>14.9h) and both C.glabrata (>40 h) isolates for all concentrations tested. The rezafungin and micafungin PAFEs could not be determined against one C.albicans isolate. Prolonged PAFE results were also noted for rezafungin (range, 18.4 to >40 h) against both C.parapsilosis isolates at all concentrations, while no micafungin PAFE or a short PAFE (range, 1.8 to 7.4h) was observed against these organisms, except at 16x bMIC. Rezafungin showed sustained growth inhibition following drug removal and displayed equivalent or longer PAFE values than micafungin against all tested Candida spp.