Disposable Cartridge Research Articles

A-343 Novel In-Line Hemolysis Detection on a Blood Gas Analyzer for Capillary Samples

Abstract Background Point-of-care (POC) blood gas and electrolyte analyzers utilizing whole blood as the predominant sample type are blinded by the presence of hemolysis until today. Moreover, capillary samples are particularly vulnerable to in-vitro hemolysis due to excessive force applied during sample collection. Here, we introduce the GEM Premier 7000 with iQM3 (GEM 7000) which incorporates automatic, in-line hemolysis detection in capillary blood gas samples without a need for additional sample volume or reagents. Methods The hemolysis detection module which is integrated into the disposable GEM cartridge uses acoustophoretic technology and photometric measurements to detect hemolysis in whole blood. In GEM 7000, hemolysis of ≥ 116 mg/dL was found to have significant interference with potassium results. A hemolysis flag is presented as the degree of hemolysis: none (0 - 50 and 51 - 115), mild (116 - 200 and 201 - 300), moderate (301 - 400), and gross (≥401). Due to volume limitations with native capillary samples and challenges in assessing hemolysis levels with a comparator assay, venous blood spiked volumetrically with hemolysate and transferred to capillary devices was used to verify the hemolysis flag performance. An agreement analysis was performed using the Roche cobas hemolysis value. The device's (GEM 7000) ability to flag hemolyzed capillary samples is demonstrated in a simulated clinical setting using native and contrived fingerstick capillary samples. Results Hemolysis verification with transferred capillary samples demonstrated that 98.2% (274/279) of samples were correctly flagged based on the cobas hemolysis value of spiked hemoglobin. Hemolysis in the native capillary samples (N = 235) was primarily distributed in the 0-50 mg/dL range. Additionally, native capillary samples altered using vigorous flea mixing (> 1 minute) showed hemolysis distribution over a wide hemolysis degree (none to gross), and their potassium results were flagged accordingly. Conclusions By providing integrated hemolysis detection in capillary blood sample measurements, the GEM Premier 7000 with iQM3 system can provide quality and confidence in the clinical interpretation of critical results such as potassium.

Optimizing equine sperm quality: an alternative to single layer centrifugation for sperm isolation.

In vitro semen purification techniques have been developed that seek to mimic the in vivo selection process in order to generate the highest possible chance of oocyte fertilization following artificial insemination. Numerous methods have been developed to isolate functional spermatozoa for artificial insemination, yet only one method, single-layer centrifugation using commercial preparations like EquiPure, has been widely employed. In this study, we have introduced a novel approach for isolating spermatozoa and compared their quality to those isolated using EquiPure. The AI port system (Memphasys, Ltd. in Sydney, Australia) features a disposable cartridge with an inoculation chamber for depositing extended semen and a harvest chamber for extracting isolated spermatozoa. These chambers are separated by a 5 µm polyethylene terephthalate (PETE) membrane, allowing highly motile spermatozoa to migrate from the inoculation chamber to the harvest chamber over a 20-minute period. This migration effectively leaves behind seminal plasma and other cell types, such as leukocytes. Comparative analyses between spermatozoa isolated with the AI port and EquiPure demonstrated that, across all measured sperm parameters, including yield, vitality, motility, morphology, DNA fragmentation, and mitochondrial superoxide generation, the AI port-isolated cells exhibited comparable or superior performance, particularly in terms of DNA fragmentation. In summary, the AI port system demonstrates the potential to efficiently isolate high-quality spermatozoa, possibly offering a cost-effective and user-friendly alternative that may enhance the success rates of artificial insemination in breeding programs. This study aimed to create a new method for refining stallion semen to increase the likelihood of a successful pregnancy through artificial insemination. While there are existing techniques for isolating high-quality sperm, the most common involves a complicated process using a centrifuge, which spins the semen to separate it. This research introduces a new approach called the AI port system that uses a disposable cartridge with two separate chambers for putting in semen and getting out isolated sperm. A membrane between the chambers acts like a filter, letting highly motile sperm swim across, leaving behind unwanted substances like bacteria and blood cells. Compared to the centrifugation method, the AI port system effectively produces sperm with comparable or better quality in various aspects, including vitality, movement, shape, DNA integrity, and energy production. In summary, the AI port system is an easy-to-use alternative with the potential to improve the success of artificial insemination in horse breeding programs.

A Deployable Viscoelastic Coagulation Monitor Enables Point-of-Care Assessment of Coagulopathy in Swine With Polytrauma.

Absence of pre-hospital coagulation tests challenges prompt management of hemostasis after trauma. The Viscoelastic Coagulation Monitor (VCM, Entegrion, Durham, NC) is a hand-held coagulation test for point-of-care. We evaluated VCM in a translational swine polytrauma model, hypothesizing that VCM correlates with a laboratory reference method, the TEG 5000 (Haemonetics, Boston, MA), and can identify coagulopathic phenotypes relevant to trauma. Our secondary hypothesis was that pre-warming of VCM disposable test cartridges using a heating plate versus pre-warming of cartridges by carrying the cartridge in the user's pocket does not significantly alter results. This study was conducted in tandem with a parent study involving anesthetized, mechanically ventilated swine (n = 20; 54 ± 5 kg) that encountered traumatic brain injury, pulmonary contusion and hemorrhage, or combination/polytrauma injury. Blood was collected at baseline, post-injury, post-shock, post-transfusion, and 6-, 24-, and 48 h post-injury to perform VCM at point-of-care. Within-group effect of time was assessed. Spearman correlation examined linear relations between VCM and standard laboratory-based coagulation tests; as well as lactate, ionized calcium, and body temperature. Logistic regression examined predictiveness of VCM to identify coagulopathic phenotypes, with receiver operator characteristic curves generated to assess diagnostic capability. At a subset of timepoints, necessity of pre-warming the VCM test cartridge using a heating plate versus pre-warming the cartridge by placement in the user's pocket was assessed by conducting simultaneous tests on two separate instruments, with results analyzed by paired t-test with crossover design. VCM revealed time-dependent changes in clotting time, clot formation time (CFT), alpha, maximum clot firmness (MCF), and lysis index (LI30). All VCM metrics correlated with the respective TEG 5000 metrics, with strongest correlation for VCM MCF with TEG MA (rhos = 0.77, P < .0001) and VCM LI30 with TEG LY30 (rhos = -0.76, P < .0001). VCM demonstrated good (area under the curve >0.70) to excellent (area under the curve >0.90) diagnostic accuracy in detection of low platelet count (MCF), low hematocrit (clotting time, clot formation time, alpha, and MCF), low fibrinogen (MCF), and high fibrinogen (alpha, MCF). There was no statistically or clinically relevant effect of cartridge warming method on results. In a trauma model, VCM detected significant changes in coagulation at point-of-care in a simplified portable form factor. VCM could enable informed hemostasis management in pre-hospital settings where coagulations tests are unavailable, pending further validation in clinical trials.

Point-of-Care Multiplex Detection of Respiratory Viruses.

The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.

Plasma drug screening using paper spray mass spectrometry with integrated solid phase extraction.

Drug overdoses have risen dramatically in recent years. We developed a simple nontargeted method using a disposable paper spray cartridge with an integrated solid phase extraction column. This method was used to screen for ~160 fentanyl analogs, synthetic cannabinoids, other synthetic drugs, and traditional drugs of abuse in over 300 authentic overdose samples collected at emergency departments in Indianapolis. A solid phase extraction step was implemented on the paper spray cartridge to enable subnanograms per milliliter synthetic drugs screening in plasma. Analysis was performed on a quadrupole orbitrap mass spectrometer using the sequential window acquisition of all theoretical fragment ion spectra approach in which tandem mass spectrometry was performed using 7 m/z isolation windows in the quadrupole. Calibration curves with isotopically labeled internal standards were constructed for 35 of the most frequently encountered synthetic and traditional illicit drugs by US toxicology labs. Additional qualitative-only drugs in a suspect screening list were also included. Limits of detection in plasma for synthetic cannabinoids ranged from 0.1 to 0.5 and 0.1 to 0.3ng/mL for fentanyl and its analogs and between 1 and 5ng/mL for most other drugs. Relative matrix effects were evaluated by determining the variation of the calibration slope in 10 different lots of biofluid and found to be between 3% and 20%. The method was validated on authentic overdose samples collected from two emergency departments in Indianapolis, Indiana, from suspected or known overdoses. Commonly detected synthetic drugs included fentanyl related substances, designer benzodiazepines such as flubromazolam, and the synthetic cannabinoid 5F-PB-22.

Abstract 6840: Automated preparation of single nuclei from FFPE samples for snRNA-Seq

Abstract S2 Genomics developed the Singulator™ 100 and 200 Systems to automate the processing of fresh, frozen, or OCT tissues into single cell or single nuclei suspensions using disposable cartridges, reagents, and customizable protocols. The systems have been used to prepare cells or nuclei from a wide range of tissues and organisms, including human, mouse, chicken, insects, snails, zebrafish, and plants. Formalin-fixed, paraffin-embedded (FFPE) tissues are the preferred format for pathologists and as many as one billion FFPE slides are in repositories. FFPE tissue samples have traditionally been difficult to study with molecular techniques due to the chemical modifications caused by the formalin fixation, which can lead to degradation and sequestration of RNA and DNA. Recent advances in single cell/nuclei RNA-Seq library preparation technology have made it possible to overcome these challenges and generate high-quality single cell or nuclei genomic data from FFPE samples. Protocols have been released for processing previously deparaffinized and rehydrated FFPE samples on the Singulator 100 or 200 into single nuclei. We now report the development of a fully automated workflow for the deparaffinization and rehydration of FFPE tissues in about 40 min, with an optional enzymatic treatment of 45 min, and processing of FFPE tissues into singulated cells or nuclei on prototype Singulator 200 systems. Data will be presented for the optimization of process parameters for cell or nuclei recovery and RNA quality for six tissues. Finally, single nuclei sequencing data will be presented for snRNA-Seq libraries from healthy and cancerous human FFPE samples including glioblastomas and colorectal cancer. These data demonstrate the successful recovery of genomic materials from FFPE samples using an automated sample preparation system for single cell/nuclei transcriptomics. Citation Format: Stevan Jovanovich, Nate Pereira, Danielle Meyer, Catherine Snopkowski, Ignas Masilionis, Brigita Meškauskaitė, Meril Takizawa, Zaki Abou-Mrad, Kenny Yu, Viviane Tabar, Kathleen Luckett, Karuna Ganesh, Nabiha Khan, Ronan Chaligne. Automated preparation of single nuclei from FFPE samples for snRNA-Seq [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 6840.

(Invited) Magnetofluidic Platforms for Point-of-Care Detection of Infectious Diseases

Commercialized rapid POC PCR technologies are either too slow, lack antimicrobial resistance data, or are too costly for widespread adoption in resource-limited settings. This talk discusses the use of magnetofluidic platforms coupled with inexpensive disposable assay cartridges to automate, accelerate, and simplify PCR tests of infectious diseases. Instead of traditional fluidic strategies for sample handling and assay automation, magnetofluidic technologies use the movement of magnetic beads through discrete droplets of reagents to capture, purify, and transfer analytes for amplification and/or detection, eliminating the need for large, complex instrumentation and fluidics typically associated with clinical laboratory PCR tests. Miniaturized and focused thermal cycling enabled by magnetofluidic PCR significantly shortens the turnaround time of a DNA/RNA test to <15 min. We demonstrated the versatile use of portable magnetofluidic platforms for various infectious disease diagnostic applications, including POC detection of sexually transmitted infections and antimicrobial susceptibility, multiplex detection of SARS-CoV-2 and respiratory pathogens, and HIV viral load testing.

Optimization of the synthesis and puri cation of 6-[18F]fluoropiperonal, synton for the preparation of complex molecules used as pet tracers

An optimized synthesis method for 6-[18F] uoropiperonal (6-[18F]FP) via nucleophilic sub itution of the nitro group in the precursor molecule (nitropiperonal, 6-NP) with [18F] uoride in the presence of tetrabutylammonium tosylate has been proposed. Using this weakly basic phase transfer catalyst, the amount of initial 6-NP in the reaction was reduced from 4.0 to 0.2 mg with negligible amounts after subsequent treatment of the reaction mixture with a strong base (potassium methoxide). In turn, this made it possible to separate 6-[18F]FP and 6-NP with similar physicochemical properties by a simple and e cient solid-phase extraction technique on disposable cartridges. 6-[18F]FP was obtained with radiochemical purity of 99% and a radiochemical yield of 10%. The content of unreacted 6-NP did not exceed 1 μg/mL, which is comparable to the results of laborious semi-preparative HPLC puri cation.

PFA-100 System: A New Method for Assessment of Platelet Dysfunction.

This is a celebratory reprint of a historical paper published in STH in 1998. The original Abstract follows.The PFA-100 system is a platelet function analyzer designed to measure platelet-related primary hemostasis. The instrument uses two disposable cartridges: a collagen/epinephrine (CEPI) and a collagen/ADP (CADP) cartridge. Previous experience has shown that CEPI cartridges detect qualitative platelet defects, including acetylsalicylic acid (ASA)-induced abnormalities, while CADP cartridges detect only thrombocytopathies and not ASA use. In this seven-center trial, 206 healthy subjects and 176 persons with various platelet-related defects, including 127 ASA users, were studied. The platelet function status was determined by a platelet function test panel. Comparisons were made as to how well the defects were identified by the PFA-100 system and by platelet aggregometry. The reference intervals for both cartridges, testing the 206 healthy subjects, were similar to values described in smaller studies in the literature (mean closure time [CT] of 132 seconds for CEPI and 93 seconds for CADP). The use of different lot numbers of cartridges or duplicate versus singleton testing revealed no differences. Compared with the platelet function status, the PFA-100 system had a clinical sensitivity of 94.9% and a specificity of 88.8%. For aggregometry, a sensitivity of 94.3% and a specificity of 88.3% were obtained. These values are based on all 382 specimens. A separate analysis of sensitivity by type of platelet defect, ASA use versus congenital thrombocytopathies, revealed for the PFA-100 system a 94.5% sensitivity in identifying ASA users and a 95.9% sensitivity in identifying the other defects. For aggregometry, the values were 100% for ASA users and 79.6% for congenital defects. Analysis of concordance between the PFA-100 system and aggregometry revealed no difference in clinical sensitivity and specificity between the systems (p > 0.9999). The overall agreement was 87.5%, with a Kappa index of 0.751. The two tests are thus equivalent in their ability to identify normal and abnormal platelet defects. Testing 126 subjects who took 325 mg ASA revealed that the PFA-100 system (CEPI) was able to detect 71.7% of ASA-induced defects with a positive predictive value of 97.8%. The overall clinical accuracy of the system, calculated from the area under the receiver operating characteristic curve, was 0.977. The data suggest that the PFA-100 system is highly accurate in discriminating normal from abnormal platelet function. The ease of operation of the instrument makes it a useful tool to use in screening patients for platelet-related hemostasis defects.

Enhancing filtration performance of submicron particle filter media through bimodal structural design

AbstractDepth filtration is a widely utilized mechanism for submicron aerosol filtration using disposable filter cartridges and facemasks. The filter media should be carefully engineered to reach high filtration efficiency and dust‐loading capacity at the expense of a low‐pressure drop (ΔP). Filter media with bimodal fiber diameter distribution enhance particle capture by creating small pores with tiny fibers, while microfibers improve airflow, reduce ΔP, and increase the effective filter area for particle retention. In this study, bimodal filters were achieved through the homogeneous distribution or layered use of nanofibers and microfibers. The impact of the bimodal design was explored using fibrous mats produced through melt‐blowing, solution‐blowing, and electroblowing methods. Keeping the basis weight of filter samples at 30 gsm, using four‐layered filters (4L) improved air permeability compared to single‐layer samples. The 4L sample exhibited the highest performance, achieving 99.52% efficiency at 148 Pa. Moreover, replacing the melt‐blown layer with bimodal mats in the 4L design increased the filtration efficiency to 99.61% keeping ΔP nearly the same. The corona discharge treatment yielded the highest efficiency (99.99%) in the 4BML sample, even after 1 month the efficiency was maintained at 99.90%, highlighting the advantage of bimodal fiber distribution in electret filters.Highlights Four‐layered filter (4L) structures resulted in improved air permeability. Bimodal layer (BL) achieved by adding SB nanofibers into the melt blowing. BL in 4L structure increased the efficiency from 99.52% to 99.61%. Modified BL sample (4BML) provides the highest QF (0.044 Pa−1) after 1 month.

Description of an In Vitro Platelet Function Analyzer-PFA-100™.

This manuscript represents a republication of a manuscript originally published in STH in 1995. This republication is to help celebrate 50 years of publishing for STH. The original abstract follows.A new in vitro system for the detection of platelet dysfunction, PFA-100®, has been developed. It provides a quantitative measure of platelet function in anticoagulated whole blood. The system comprises a microprocessor-controlled instrument and a disposable test cartridge containing a biologically active membrane. The instrument aspirates a blood sample under constant vacuum from the sample reservoir through a capillary and a microscopic aperture cut into the membrane. The membrane is coated with collagen and epinephrine or adenosine 5'-diphosphate. The presence of these biochemical stimuli, and the high shear rates generated under the standardized flow conditions, result in platelet attachment, activation, and aggregation, slowly building a stable platelet plug at the aperture. The time required to obtain full occlusion of the aperture is reported as the "closure time." We have found that impairment of von Willebrand factor, or inhibition of platelet receptors glycoprotein Ib or IIb/IIIa with monoclonal antibodies or peptides, resulted in abnormal closure times. An antifibrinogen antibody, in contrast, failed to show any effect. The test appears to be sensitive to platelet adherence and aggregation abnormalities. The PFA-100® system has potential applications in routine evaluation of platelet function in the clinical setting because of its accuracy, case of operation, and rapid turnaround of results.

Highly Sensitive Chip-Based Digital PCR Platform for Quantitative Detection of BCR::ABL1 Transcripts throughout CML Treatment

Introduction: With various BCR::ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable disease and current therapeutic goal is treatment-free remission (TFR). To achieve a successful TFR in more patients, a precise assessment of minimal residual disease (MRD) is one of the prerequisite requirements. Since undetectable BCR::ABL1 fusion transcripts by conventional Taqman™-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of the CML clone, it requires the development of more sensitive technologies. Currently, to overcome some limitations of conventional qPCR, such as rigorous standardization process and variations in sensitivity between laboratories, digital quantitative-PCR (dqPCR) methods have been developed for accurate and precise detection and its use in the routine clinical practice is gradually expanding. However, current dqPCR methods require the inclusion of one or more negative partitions to calculate the concentration using Poisson distribution. Consequently, this leads to a more than a 10-fold decrease in the maximum detectable concentration in a single test compared to conventional qPCR. Therefore, we have developed a novel technology, High Dynamic Range Digital Real-time PCR (so called HDR-drPCR) to achieve wider dynamic range, which allows more stable, accurate and sensitive monitoring of BCR::ABL1 in a timely manner. Methods: For linearity assessment, we used BCR::ABL1 positive (e14a2) K562 cell line diluted to 7 points by 10-fold serial dilutions from 25 to 2.5x10 -5 %IS with BCR::ABL1 negative HL60 cells. In HDR-drPCR, the GUSB gene was employed as a control gene to determine %IS values. For this purpose, the 1 st WHO international genetic reference panel was utilized to calculate the correction factor (CF). With the results from cell lines, we also evaluated equivalence between conventional qPCR and HDR-drPCR using 219 clinical specimens. Among them, 100 samples were determined as DMR (&amp;lt;0.01%IS) based on the qPCR test results In detail, conventional qPCR was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, USA) for cDNA synthesis and Real-Q BCR-ABL Quantification Kit (Biosewoom, South Korea) for qPCR. For HDR-drPCR (1-step reverse transcriptase PCR), 10 6 to 10 7 copies per reaction of GUSB gene were applied to disposable cartridges of Genotizer™ HDR BCR::ABL1 Major IS detection kit (Optolane Technologies, South Korea) with LOAA platform (Optolane Technologies, Korea). Based on the results, correlation analysis between qPCR and HDR-drPCR was performed using correlation graphs and Blend-Altman plots. Results: Linearity was assessed by the dilution of RNA of K562 with RNA of HL60 using 10-fold serial dilution of up to - 7 log. The result showed accurate linearity (R 2 = 0.9972) at the range of BCR::ABL1/GUSB ratio of 10 1 to 10 -5(%). Notably, HDR-drPCR technology allowed to detect the level of BCR::ABL1 transcript of MR 6.34 (0.00005 %IS) (Figure 1A). The correlation analysis for 209 clinical samples showed that measured values from both qPCR and HDR-drPCR were closely correlated with the R 2 of 0.973 1 (Figure 1B) . The Bland-Altman plot showed a mean bias of 0.159, and the limit of agreement at the 95% confidence interval (LOA95) was calculated as -0.387 to 0.705. Furthermore, the concordance rate between the two methods was 96.17%, as measured by the proportion of samples in which the difference between the two methods was within a factor of 5. This indicates a strong concordance between the two methods. HDR-drPCR reliably detected BCR::ABL1 transcripts in all 90 clinical specimens measured below 0.01%IS (DMR) using qPCR. Additionally, HDR-drPCR detected trace amounts of BCR::ABL1 transcripts in 6 out of 10 specimens that were not detected by qPCR, showcasing its improved sensitivity compared to the conventional qPCR method. Conclusion: Taken together, we believe our high dynamic range real-time digital PCR(HDR-drPCR) may minimize false positive results and maximize the analytical sensitivity of detecting BCR::ABL1 fusion transcripts and thus, would be useful for accurately assessing treatment in routine clinical practice and facilitating the application of treatment-free remission (TFR). We will include these results in further intensive studies focusing on DMR clinical specimens Study support: Funded by Korea Research Foundation

A novel tailed primer nucleic acid test for detection of HPV 16, 18 and 45 DNA at the point of care

Cervical cancer is a leading cause of death for women in low-resource settings despite being preventable through human papillomavirus (HPV) vaccination, early detection, and treatment of precancerous lesions. The World Health Organization recommends high-risk HPV (hrHPV) as the preferred cervical cancer screening strategy, which is difficult to implement in low-resource settings due to high costs, reliance on centralized laboratory infrastructure, and long sample-to-answer times. To help meet the need for rapid, low-cost, and decentralized cervical cancer screening, we developed tailed primer isothermal amplification and lateral flow detection assays for HPV16, HPV18, and HPV45 DNA. We translated these assays into a self-contained cartridge to achieve multiplexed detection of three hrHPV genotypes in a disposable cartridge. The developed test achieves clinically relevant limits of detection of 50–500 copies per reaction with extracted genomic DNA from HPV-positive cells. Finally, we performed sample-to-answer testing with direct lysates of HPV-negative and HPV-positive cell lines and demonstrated consistent detection of HPV16, HPV18, and HPV45 with 5000–50,000 cells/mL in < 35 min. With additional optimization to improve cartridge reliability, incorporation of additional hrHPV types, and validation with clinical samples, the assay could serve as a point-of-care HPV DNA test that improves access to cervical cancer screening in low-resource settings.

Optimization of the Synthesis and Purification of 6-[18F]Fluoropiperonal, Synthon for the Preparation of Complex Molecules Used as PET Tracers

An optimized synthesis method for 6-[18F]fluoropiperonal (6-[18F]FP) via nucleophilic substitution of the nitro group in the precursor molecule (nitropiperonal, 6-NP) with [18F]fluoride in the presence of tetrabutylammonium tosylate has been proposed. Using this weakly basic phase transfer catalyst, the amount of initial 6-NP in the reaction was reduced from 4.0 to 0.2 mg with negligible amounts after subsequent treatment of the reaction mixture with a strong base (potassium methoxide). In turn, this made it possible to separate 6-[18F]FP and 6-NP with similar physicochemical properties by a simple and efficient solid-phase extraction technique on disposable cartridges. 6-[18F]FP was fabricated with 99% radiochemical purity and a radiochemical yield of 10%. The content of unreacted 6-NP did not exceed 1 µg/mL, which is comparable to the results of laborious semi-preparative HPLC purification.

B-236 Portable 2 Hour Sample-to-Answer Multi-pathogen Phenotypic Antibiotic Susceptibility Test (AST)

Abstract Background Antimicrobial Resistance is a rapidly growing issue worldwide, arising from the overuse and misuse of antibiotics. The strongest tool the medical community has available to combat AMR is rapid, correct application of antibiotics to identified bacterial infections. Clinics and physician offices seek on-site rapid solutions to speed the pathway to timely and appropriate antibiotic therapies. GeneCapture is prototyping a rapid, sample-to-answer product to provide antibiotic susceptibility results directly from infected urine or wound samples. The test provides a susceptible vs non-susceptible call in under two hours and can be applied to samples with single or mixed pathogens. With support from the Defense Health Agency, a portable table-top instrument and disposable cartridge are being engineered to run this test at the point of care, allowing same-day prescriptions. The process is currently operational on the lab bench. Methods Fluorescent labeling of bacterial species, followed by optical counting, determines population growth with/without a suite of antibiotics. An on-board algorithm instantly converts raw cell numbers into a differential call of Susceptible or Non-susceptible for each bug/drug pair. A disposable cartridge with on-board antibiotics has been designed and is being finalized for fabrication. The optical counting is operating in a desktop device. Results Testing to date includes 124 strains from 7 of the most common Urinary Tract Infection and wound pathogens against a panel of six antibiotics. A total of 541 antibiotic/bacterial strain pairs in single or multiple pathogen antibiotic susceptibility testing (AST), are producing an initial 88.7% agreement to gold standard culture results. These results are all generated in two hours or less of assay time and represent a strong preliminary data set. Conclusion This 2-hour portable, novel, phenotypic AST profiling will produce informed and effective treatment options for medics, clinics and physician offices long before existing AST methods could return results.

Single-Molecule Bioelectronic Portable Array for Early-Diagnosis of Pancreatic Cancer Precursors.

A cohort of 47 patients is screened for pancreatic cancer-precursors with a portable 96-well bioelectronic sensing-array for single-molecule assay in cysts fluid and blood plasma deployable at point-of-care (POC). Pancreatic cancer precursors are mucinous cysts diagnosed with a sensitivity of at most 80% by state-of-the-art cytopathological molecular analyses (e.g., KRASmut DNA). Adding the simultaneous assay of proteins related to malignant transformation (e.g., MUC1 and CD55) is deemed essential to enhance diagnostic accuracy. The bioelectronic array here proposed, based on the SiMoT (Single-Molecule-with-a-large-Transistor) technology, can assay both nucleic acids and proteins at the single-molecule Limit-Of-Identification, LOI (<1% of false-positives and false-negatives). It comprises an ELISA-like 8×12-array organic-electronics disposable cartridge with an electrolyte-gated organic transistor sensor array, and a reusable reader, integrating a custom Si-IC chip, operating via software installed on a USB-connected smart device. The cartridge is complemented by a 3D-printed sensing gate cover plate. KRASmut , MUC1, and CD55 biomarkers either in plasma or cysts-fluid from 5-6 patients at a time, are multiplexed at single-molecule LOI in 1.5 hours. The pancreatic cancer precursors are classified via a machine-learning analysis resulting in at least 96% diagnostic-sensitivity and 100% diagnostic-specificity. This preliminary study opens the way to POC liquid-biopsy-based early diagnosis of pancreatic-cancer precursors in plasma. This article is protected by copyright. All rights reserved.

P24 Point of care rapid urinary tract infection phenotypic antibiotic susceptibility test

Abstract Background Urinary tract infections (UTIs) are one of the most common infections in the world today. In the last few years, the occurrence of antimicrobial resistant pathogens causing UTIs has rapidly increased. One in three UTIs are trimethoprim/sulfamethoxazole (Bactrim) resistant, and one in five are resistant to other common antibiotic choices. This growing resistance is mainly due to overuse or misuse of antibiotics. The strongest tool the medical community has available to combat antimicrobial resistance (AMR) is rapid, correct application of antibiotics to an identified bacterial infection. Clinics and physician offices seek on-site rapid solutions to speed the pathway to timely and appropriate antibiotic therapies. GeneCapture is developing products that perform rapid UTI pathogen identification and antimicrobial susceptibility tests (ASTs) directly from infected urine samples. The AST provides a susceptible versus non-susceptible call in under 2 h and can be applied to samples with single or mixed pathogens. A portable tabletop instrument and disposable cartridge are being engineered to run this test at the point of care, allowing same-day prescriptions. The process is currently operational on the lab bench. Methods Fluorescent labelling of bacterial species, followed by optical counting, determines population growth with/without a suite of antibiotics. An on-board algorithm instantly converts raw cell numbers into a differential call of susceptible or non-susceptible for each bug/drug pair. A disposable cartridge with on-board antibiotics has been designed and is being finalized for fabrication. The optical counting is operating in a desktop device. Results Testing to date includes 124 strains from 7 of the most common UTI and wound pathogens against a panel of six antibiotics. A total of 541 antibiotic/bacterial strain pairs in single or multiple pathogen antibiotic susceptibility testing produce an initial 88.7% agreement to gold standard culture results. These results are all generated in 2 h or less of assay time and represent a strong preliminary dataset. Conclusions This 2 h portable, novel, phenotypic AST profiling will produce informed and effective treatment options for medics, clinics and physician offices long before existing AST methods could return results.

Multicenter evaluation of a simple and sensitive nucleic acid self-testing for SARS-CoV-2

A rapid and accurate COVID-19 diagnosis is a prerequisite for blocking the source of infection as soon as possible and taking the appropriate medical action. Herein, we developed GeneClick, a device for nucleic acid self-testing of SARS-CoV-2, consisting of three modules: a sampling kit, a microfluidic chip-based disposable cartridge, and an amplification reader. In addition, we evaluated the clinical performance of GeneClick using 2162 nasal swabs collected at three medical institutions, using three commercial RT-qPCR kits and an antigen self-test as references. Compared to RT-qPCR, the sensitivity and specificity of the GeneClick assay were 97.93% and 99.72%, respectively, with a kappa value of 0.979 (P ​< ​0.01). Of the 2162 samples, 2076 were also tested for SARS-CoV-2 antigens. Among the 314 positive samples identified by GeneClick assay, 63 samples were undetected by antigen tests. Overall, the GeneClick nucleic acid self-test demonstrated higher accuracy than the antigen-based detection. Based on the additional features, including simple operation, affordable price, portable device, and reliability of smartphone APP-driven sampling and result reporting, GeneClick offers a powerful tool for field-based SARS-CoV-2 detection in primary healthcare institutions or at-home use.

High-throughput microbead assay system with a portable, cost-effective Wi-Fi imaging module, and disposable multi-layered microfluidic cartridges for virus and microparticle detection, and tracking.

In recent years biomedical scientific community has been working towards the development of high-throughput devices that allow a reliable, rapid and parallel detection of several strains of virus or microparticles simultaneously. One of the complexities of this problem lies on the rapid prototyping of new devices and wireless rapid detection of small particles and virus alike. By reducing the complexity of microfluidics microfabrication and using economic materials along with makerspace tools (Kundu et al. 2018) it is possible to provide an affordable solution to both the problems of high-throughput devices and detection technologies. We present the development of a wireless, standalone device and disposable microfluidics chips that rapidly generate parallel readouts for selected, possible virus variants from a nasal or saliva sample, based on motorized and non-motorized microbeads detection, and imaging processing of the motion tracks of these beads in micrometers. Microbeads and SARS-CoV-2 COVID-19 Delta variant were tested as proof-of-concept for testing the microfluidic cartridges and wireless imaging module. The Microbead Assay (MA) system kit consists of a Wi-Fi readout module, a microfluidic chip, and a sample collection/processing sub-system. Here, we focus on the fabrication and characterization of the microfluidic chip to multiplex various micrometer-sized beads for economic, disposable, and simultaneous detection of up to six different viruses, microparticles or variants in a single test, and data collection using a commercially available, Wi-Fi-capable, and camera integrated device (Fig. 1).

Application of Shear Horizontal Surface Acoustic Wave (SH-SAW) Immunosensor in Point-of-Care Diagnosis.

Point-of-care testing (POCT), also known as on-site or near-patient testing, has been exploding in the last 20 years. A favorable POCT device requires minimal sample handling (e.g., finger-prick samples, but plasma for analysis), minimal sample volume (e.g., one drop of blood), and very fast results. Shear horizontal surface acoustic wave (SH-SAW) biosensors have attracted a lot of attention as one of the effective solutions to complete whole blood measurements in less than 3 min, while providing a low-cost and small-sized device. This review provides an overview of the SH-SAW biosensor system that has been successfully commercialized for medical use. Three unique features of the system are a disposable test cartridge with an SH-SAW sensor chip, a mass-produced bio-coating, and a palm-sized reader. This paper first discusses the characteristics and performance of the SH-SAW sensor system. Subsequently, the method of cross-linking biomaterials and the analysis of SH-SAW real-time signals are investigated, and the detection range and detection limit are presented.