Automated wash and reuse of disposable pipette tips in a SARS-CoV-2 RT-qPCR diagnostic pipeline.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic led to global shortages in laboratory consumables, in particular for automated PCR. The Technical University of Denmark supported Danish hospitals from 2020 to 2022, conducting SARS-CoV-2 RT-qPCR on around 10,000 patient samples daily. We encountered shortages of disposable pipette tips used with automated liquid handlers that transferred oropharyngeal swab samples to 96-well microplates before RNA extraction. To enable tip reuse, we developed an automated protocol for washing tips with a 0.5 % sodium hypochlorite solution. This effectively eliminated carry-over of genomic material and the wash solution remained effective when stored in an open reservoir at ambient temperatures for 24 h. A three-day validation setup demonstrated the robustness of the tip wash protocol. Reducing the number of tips used for transferring samples to 96-well microplates from 96 to 8 enabled us to mitigate pipette tip shortages, lower costs, and minimize plastic waste generation.

Comparison of Otsu and an adapted Chan-Vese method to determine thyroid active volume using Monte Carlo generated SPECT images.

BACKGROUND: The Otsu method and the Chan-Vese model are two methods proven to perform well in determining volumes of different organs and specific tissue fractions. This study aimed to compare the performance of the two methods regarding segmentation of active thyroid gland volumes, reflecting different clinical settings by varying the parameters: gland size, gland activity concentration, background activity concentration and gland activity concentration heterogeneity. METHODS: A computed tomography was performed on three playdough thyroid phantoms with volumes 20, 35 and 50 ml. The image data were separated into playdough and water based on Hounsfield values. Sixty single photon emission computed tomography (SPECT) projections were simulated by Monte Carlo method with isotope Technetium-99 m ([Formula: see text]Tc). Linear combinations of SPECT images were made, generating 12 different combinations of volume and background: each with both homogeneous thyroid activity concentration and three hotspots of different relative activity concentrations (48 SPECT images in total). The relative background levels chosen were 5 %, 10 %, 15 % and 20 % of the phantom activity concentration and the hotspot activities were 100 % (homogeneous case) 150 %, 200 % and 250 %. Poisson noise, (coefficient of variation of 0.8 at a 20 % background level, scattering excluded), was added before reconstruction was done with the Monte Carlo-based SPECT reconstruction algorithm Sahlgrenska Academy reconstruction code (SARec). Two different segmentation algorithms were applied: Otsu's threshold selection method and an adaptation of the Chan-Vese model for active contours without edges; the results were evaluated concerning relative volume, mean absolute error and standard deviation per thyroid volume, as well as dice similarity coefficient. RESULTS: Both methods segment the images well and deviate similarly from the true volumes. They seem to slightly overestimate small volumes and underestimate large ones. Different background levels affect the two methods similarly as well. However, the Chan-Vese model deviates less and paired t-testing showed significant difference between distributions of dice similarity coefficients (p-value [Formula: see text]). CONCLUSIONS: The investigations indicate that the Chan-Vese model performs better and is slightly more robust, while being more challenging to implement and use clinically. There is a trade-off between performance and user-friendliness.

A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis.

BACKGROUND: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. RESULTS: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. CONCLUSION: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.

Autoradiography and biopsy measurements of a resected hepatocellular carcinoma treated with 90 yttrium radioembolization demonstrate large absorbed dose heterogeneities.

PURPOSE: Radioembolization is an alternative palliative treatment for hepatocellular carcinoma. Here, we examine the uptake differences between tumor tissue phenotypes and present a cross-section of the absorbed dose throughout a liver tissue specimen. METHODS AND MATERIALS: A patient with hepatocellular carcinoma was treated with 90Y radioembolization followed by liver tissue resection. Gamma camera images and autoradiographs were collected and biopsy tissue samples were analyzed using a gamma well counter and light microscopy. RESULTS: An analysis of 25 punched biopsy tissue samples identified 4 tissue regions: Normal tissue, viable tumor tissue with and without infarcted areas, and tumor areas with postnecrotic scar tissue. Autoradiography and biopsy tissue sample measurements showed large dose differences between viable and postnecrotic tumor tissue (159 Gy vs 23 Gy). CONCLUSIONS: Radioembolization of 90 yttrium with resin microspheres produces heterogeneous-absorbed dose distributions in the treatment of unifocal hepatic malignancies that could not be accurately determined with current gamma camera imaging techniques.

Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR.

Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 1011 molecules/mm2. In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/µl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.

Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection.

Simulation Model of Microsphere Distribution for Selective Internal Radiation Therapy Agrees With Observations.

PURPOSE: To perform a detailed analysis of microsphere distribution in biopsy material from a patient treated with (90)Y-labeled resin spheres and characterize microsphere distribution in the hepatic artery tree, and to construct a novel dichotomous bifurcation model for microsphere deposits and evaluate its accuracy in simulating the observed microsphere deposits. METHODS AND MATERIALS: Our virtual model consisted of arteries that successively branched into 2 new generations of arteries at 20 nodes. The artery diameter exponentially decreased from the lowest generation to the highest generation. Three variable parameters were optimized to obtain concordance between simulations and measure microsphere distributions: an artery coefficient of variation (ACV) for the diameter of all artery generations and the microsphere flow distribution at the nodes; a hepatic tree distribution volume (HDV) for the artery tree; and an artery diameter reduction (ADR) parameter. The model was tested against previously measured activity concentrations in 84 biopsies from the liver of 1 patient. In 16 of 84 biopsies, the microsphere distribution regarding cluster size and localization in the artery tree was determined via light microscopy of 30-µm sections (mean concentration, 14 microspheres/mg; distributions divided into 3 groups with mean microsphere concentrations of 4.6, 14, and 28 microspheres/mg). RESULTS: Single spheres and small clusters were observed in terminal arterioles, whereas large clusters, up to 450 microspheres, were observed in larger arterioles. For 14 microspheres/mg, the optimized parameter values were ACV=0.35, HDV = 50 cm(3), and ADR=6 µm. For 4.6 microspheres/mg, ACV and ADR decreased to 0.26 and 0 µm, respectively, whereas HDV increased to 130 cm(3). The opposite trend was observed for 28 microspheres/mg: ACV = 0.49, HDV = 20 cm(3), and ADR = 8 µm. CONCLUSION: Simulations and measurements reveal that microsphere clusters are larger and more common in volumes with high microsphere concentrations and indicate that the spatial distribution of the artery tree must be considered in estimates of microsphere distributions.

Increased absorbed liver dose in Selective Internal Radiation Therapy (SIRT) correlates with increased sphere-cluster frequency and absorbed dose inhomogeneity.

BACKGROUND: The higher tolerated mean absorbed dose for selective internal radiation therapy (SIRT) with intra-arterially infused (90)Y microspheres compared to external beam therapy is speculated to be caused by absorbed dose inhomogeneity, which allows for liver regeneration. However, the complex liver microanatomy and rheology makes modelling less valuable if the tolerance doses are not based on the actual microsphere distribution. The present study demonstrates the sphere distribution and small-scale absorbed dose inhomogeneity and its correlation with the mean absorbed dose in liver tissue resected after SIRT. METHODS: A patient with marginally resectable cholangiocarcinoma underwent SIRT 9 days prior to resection including adjacent normal liver tissue. The resected specimen was formalin-fixed and sliced into 1 to 2-mm sections. Forty-one normal liver biopsies 6-8 mm in diameter were punched from these sections and the radioactivity measured. Sixteen biopsies were further processed for detailed analyses by consecutive serial sectioning of 15 30-µm sections per biopsy, mounted and stained with haematoxylin-eosin. All sections were scrutinised for isolated or conglomerate spheres. Small-scale dose distributions were obtained by applying a (90)Y-dose point kernel to the microsphere distributions. RESULTS: A total of 3888 spheres were found in the 240 sections. Clusters were frequently found as strings in the arterioles and as conglomerates in small arteries, with the largest cluster comprising 453 spheres. An increased mean absorbed dose in the punch biopsies correlated with large clusters and a greater coefficient of variation. In simulations the absorbed dose was 5-1240 Gy; 90% were 10-97 Gy and 45% were <30 Gy, the assumed tolerance in external beam therapy. CONCLUSIONS: Sphere clusters were located in both arterioles and small arteries and increased in size with increasing sphere concentration, resulting in increased absorbed dose inhomogeneity, which contradicts earlier modelling studies.

Heterogeneity of microsphere distribution in resected liver and tumour tissue following selective intrahepatic radiotherapy.

BACKGROUND: Selective arterial radioembolisation of liver tumours has increased, because of encouraging efficacy reports; however, therapeutic parameters used in external beam therapy are not applicable for understanding and predicting potential toxicity and efficacy, necessitating further studies of the physical and biological characteristics of radioembolisation. The aim was to characterise heterogeneity in the distribution of microspheres on a therapeutically relevant geometric scale considering the range of yttrium-90 ((90)Y) ß-particles. METHODS: Two patients with intrahepatic cholangiocarcinoma, marginally resectable, were treated by selective arterial embolisation with (90)Y resin microspheres (SIRTEX®), followed 9 days post-infusion by resection, including macroscopic tumour tissue and surrounding normal liver parenchyma. Formalin-fixed, sectioned resected tissues were exposed to autoradiographic films, or tissue biopsies of various dimensions were punched out for activity measurements and microscopy. RESULTS: Autoradiography and activity measurements revealed a higher activity in tumour tissue compared to normal liver parenchyma. Heterogeneity in activity distribution was evident in both normal liver and tumour tissue. Activity measurements were analysed in relation to the sample mass (5 to 422 mg), and heterogeneities were detected by statistical means; the larger the tissue biopsies, the smaller was the coefficient of variation. The skewness of the activity distributions increased with decreasing biopsy mass. CONCLUSIONS: The tissue activity distributions in normal tissue were heterogeneous on a relevant geometric scale considering the range of the ionising electrons. Given the similar and repetitive structure of the liver parenchyma, this finding could partly explain the tolerance of a relatively high mean absorbed dose to the liver parenchyma from ß-particles.

Pre-storage of gelified reagents in a lab-on-a-foil system for rapid nucleic acid analysis.

Reagent pre-storage in a microfluidic chip can enhance operator convenience, simplify the system design, reduce the cost of storage and shipment, and avoid the risk of cross-contamination. Although dry reagents have long been used in lateral flow immunoassays, they have rarely been used for nucleic acid-based point-of-care (POC) assays due to the lack of reliable techniques to dehydrate and store fragile molecules involved in the reaction. In this study, we describe a simple and efficient method for prolonged on-chip storage of PCR reagents. The method is based on gelification of all reagents required for PCR as a ready-to-use product. The approach was successfully implemented in a lab-on-a-foil system, and the gelification process was automated for mass production. Integration of reagents on-chip by gelification greatly facilitated the development of easy-to-use lab-on-a-chip (LOC) devices for fast and cost-effective POC analysis.

Radiation exposure during liver surgery after treatment with (90)Y microspheres, evaluated with computer simulations and dosimeter measurements.

PURPOSE: Two patients with liver tumours were planned for a combined treatment, including surgery with preceding injections of ß(-) radiation emitting (90)Y microspheres (SIRTEX(®)). The aim of this paper is to present a method of pre-surgical computer simulations of the absorbed dose rate on the surface of tumour tissue, combined with measurements of the actual absorbed dose rate on resected tissue, in order to estimate the absorbed dose to a surgeon's fingers during such surgery procedures. METHODS AND MATERIALS: The dose rates from ß(-) radiation on the surface of tumour tissue were simulated with the software VARSKIN(®) Mod 2. The activity concentrations in tumours were estimated, based on SPECT/CT distribution studies of (99m)Tc-MAA and confirmed by SPECT/CT bremsstrahlung studies of (90)Y microspheres. The activity distributions were considered as homogeneous within the tumour regions. The absorbed dose rates at different tumour tissue spots were calculated based on measurements with thermo-luminescent dosimeters (TLD) fastened on resected tissue. RESULTS: The simulations showed a good agreement with the averaged absorbed dose rates based on TLD measurements performed on resected tissue, differing by 13% and 4% respectively. The absorbed dose rates at the measured maximum hotspots were twice as high as the average dose rates for both patients. CONCLUSION: The data is not sufficient in order to draw any general conclusions about dose rates on tumour tissue during similar surgeries, neither about the influence of dose rate heterogeneities nor about average dose rates. However, the agreement between simulations and measurements on these limited data indicate that this approach is a promising method for estimations of the radiation exposure to the surgeons' fingers during this kind of surgery procedure. More data from similar surgeries are necessary in order to validate the method.

Gold Nanoparticles-Coated SU-8 for Sensitive Fluorescence-Based Detections of DNA.

SU-8 epoxy-based negative photoresist has been extensively employed as a structural material for fabrication of numerous biological microelectro-mechanical systems (Bio-MEMS) or lab-on-a-chip (LOC) devices. However, SU-8 has a high autofluorescence level that limits sensitivity of microdevices that use fluorescence as the predominant detection workhorse. Here, we show that deposition of a thin gold nanoparticles layer onto the SU-8 surface significantly reduces the autofluorescence of the coated SU-8 surface by as much as 81% compared to bare SU-8. Furthermore, DNA probes can easily be immobilized on the Au surface with high thermal stability. These improvements enabled sensitive DNA detection by simple DNA hybridization down to 1 nM (a two orders of magnitude improvement) or by solid-phase PCR with sub-picomolar sensitivity. The approach is simple and easy to perform, making it suitable for various Bio-MEMs and LOC devices that use SU-8 as a structural material.

Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay.

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.

A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR.

The endemic of Avian Influenza Virus (AIV) in Asia and epizootics in some European regions have caused serious economic losses. Multiplex reverse-transcriptase (RT) PCR has been developed to detect and subtype AIV. However, the number of targets that can be amplified in a single run is limited because of uncontrollable primer-primer interferences. In this paper, we describe a lab-on-a-chip device for fast AIV screening by integrating DNA microarray-based solid-phase PCR on a microfluidic chip. A simple UV cross-linking method was used to immobilize the DNA probes on unmodified glass surface, which makes it convenient to integrate microarray with microfluidics. This solid-phase RT-PCR method combined RT amplification of extracted RNA in the liquid phase and species-specific nested PCR on the solid phase. Using the developed approach, AIV viruses and their subtypes were unambiguously identified by the distinct patterns of amplification products. The whole process was reduced to less than 1 hour and the sample volume used in the microfluidic chip was at least 10 times less than in the literature. By spatially separating the primers, highly multiplexed amplification can be performed in solid-phase PCR. Moreover, multiplex PCR and sequence detection were done in one step, which greatly simplified the assay and reduced the processing time. Furthermore, by incorporating the microarray into a microchamber-based PCR chip, the sample and the reagent consumption were greatly reduced, and the problems of bubble formation and solution evaporation were effectively prevented. This microarray-based PCR microchip can be widely employed for virus detection and effective surveillance in wild avian and in poultry productions.

Detection of avian influenza virus by fluorescent DNA barcode-based immunoassay with sensitivity comparable to PCR.

In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection involves the sandwiching of the target AIV between magnetic immunoprobes and barcode-carrying immunoprobes. Because each barcode-carrying immunoprobe is functionalized with a multitude of fluorophore-DNA barcode strands, many DNA barcodes are released for each positive binding event resulting in amplification of the signal. Using an inactivated H16N3 AIV as a model, a linear response over five orders of magnitude was obtained, and the sensitivity of the detection was comparable to conventional RT-PCR. Moreover, the entire detection required less than 2 hr. The results indicate that the method has great potential as an alternative for surveillance of epidemic outbreaks caused by AIV, other viruses and microorganisms.