New Concept and Apparatus for Cytocentrifugation and Cell Processing for Microscopy Analysis.

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.

Enrichment of leukocytes in peripheral blood using 3D printed tubes.

Leukocytes have an essential role in patient clinical trajectories and progression. Traditional methods of leukocyte enrichment have many significant limitations for current applications. It is demonstrated a novel 3D printing leukocyte sorting accumulator that combines with centrifugation to ensure label-free initial leukocyte enrichment based on cell density and size. The internal structure of leukocyte sorting accumulator (revealed here in a new design, leukocyte sorting accumulator-3, upgraded from earlier models), optimizes localization of the buffy coat fraction and the length of the period allocated for a second centrifugation step to deliver a higher recovery of buffy coats than earlier models. Established methodological parameters were evaluated for reliability by calculating leukocyte recovery rates and erythrocyte depletion rates by both pushing and pulling methods of cell displacement. Results indicate that leukocyte sorting accumulator-3 achieves a mean leukocytes recovery fraction of 96.2 ± 2.38% by the pushing method of layer displacement. By the pulling method, the leukocyte sorting accumulator-3 yield a mean leukocytes recovery fraction of 94.4 ± 0.8%. New procedures for preliminary enrichment of leukocytes from peripheral blood that avoid cellular damage, as well as avert metabolic and phase cycle intervention, are required as the first step in many modern clinical and basic research assays.

Performance of ivisen IA-1400, a new point-of-care device with an internal centrifuge system, for the measurement of cardiac troponin I levels.

BACKGROUND: We present the analytical performance of the ivisen IA-1400, a new point-of-care device that features a characteristic built-in centrifuge system, to measure blood cardiac troponin I (cTnI) levels. METHODS: Whole blood and plasma samples obtained from patients who visited Korea University Guro Hospital were used to analyze measurement range, cross-reactivity, interference, and sensitivity and specificity. We performed a correlation analysis of the ivisen IA-1400 versus the Access AccuTnI+3 immunoassay using the UniCel™ DxI 800 platform and the PATHFAST™ hs-cTnI assay. RESULTS: Within-run precisions were classified as low, 9.8%; middle, 10.2%; and high, 8.5%. The limit of blank was 3.1 ng/L for plasma samples and 4.3 ng/L for whole blood samples. The limit of detection was 8.4 ng/L for plasma samples and 10.0 ng/L for whole blood samples, respectively. The limit of quantitation at a coefficient of variation of 20% and 10% was 19.5 ng/L and 45.5 ng/L for plasma samples, respectively. The comparative evaluation between the two other assays and ivisen IA-1400 showed excellent correlation, with Spearman's correlation coefficients (R) of 0.992 and 0.985. The sensitivity and specificity of ivisen IA-1400 using the optimum cut-off value of 235 ug/L were 94.6% and 98.2%, respectively. CONCLUSION: The ivisen IA-1400 showed acceptable and promising performance in cTnI measurements using whole blood and plasma samples, with limited information in the clinical performance. The flexibility for sample selection using the internal centrifugation system is the main advantage of this point-of-care device.

Multiple authentications of high-value milk by centrifugal microfluidic chip-based real-time fluorescent LAMP.

Adulteration of food ingredients, particularly replacement of high-value milk with low-cost milk, affects food safety. For rapid and accurate identification of the possible adulterating milk species in an unknown sample, a centrifugal microfluidic chip-based real-time fluorescent multiplex loop-mediated isothermal amplification (LAMP) assay was developed to simultaneously detect milk from cow, camel, horse, goat, and yak. Using precoated primers in different reaction wells, the centrifugal microfluidic chip markedly simplified the detection process and reduced false-positive results. The entire amplification was completed within 90 min with a genomic detection limit of 0.05 ng/µL in cow, camel, horse, and goat milk and 0.005 ng/µL in yak milk. Using simulated adulterated samples for validation, the detection limit for adulterated milk samples was 2.5%, satisfying authentication requirements, as the proportion of adulterated milk higher than 10% affects economic interests. Therefore, this simple, centrifugal, microfluidic chip-based multiplex real-time fluorescent LAMP assay can simultaneously detect common milk species in commercial products to enable accurate labeling.

A novel modified thaw-siphon method for extracting cryoprecipitate: The Bokutoh-siphon method.

BACKGROUND: Cryoprecipitate (CRYO) is neither produced nor supplied by the Japanese Red Cross Society. A novel CRYO extraction method established in-house by modifying a thaw-siphon technique was demonstrated in this study. STUDY DESIGN AND METHODS: A pack of fresh frozen plasma was thawed and equally divided into two bags for CRYO extraction by different methods. CRYO was extracted from the blood plasma using a standard centrifugation method and our modified thaw-siphon method (Bokutoh-siphon method; B method). The two different CRYOs extracted were analyzed to compare the differences in the amount of fibrinogen recovered, clotting factors extracted, and clotting activity. RESULTS: The amount of fibrinogen in the CRYO extracted using the B-siphon method was similar to that obtained using the standard method (recovery of fibrinogen: B-siphon method: 71.2% vs. standard method: 61.0%). The amount of clotting XIII factor extracted using the B-siphon method was significantly lower than those extracted using the standard method. On the other hand, clotting II, V factors, and C1q esterase inhibitor not concentrated in CRYO content from the B-siphon method were significantly higher than that from the standard method. CONCLUSION: A new in-house CRYO preparation method was established by modifying a previously used thaw-siphon method. A coagulation factor-rich CRYO was extracted from plasma frozen at -40°C along with the first fraction of thawed plasma, without using a large-capacity refrigerated centrifuge for blood bags.

Rapid molecular diagnostics of COVID-19 by RT-LAMP in a centrifugal polystyrene-toner based microdevice with end-point visual detection.

Infection caused by the new coronavirus (SARS-CoV-2) has become a serious worldwide public health problem, and one of the most important strategies for its control is mass testing. Loop-mediated isothermal amplification (LAMP) has emerged as an important alternative to simplify the diagnostics of infectious diseases. In addition, an advantage of LAMP is that it allows for easy reading of the final result through visual detection. However, this step must be performed with caution to avoid contamination and false-positive results. LAMP performed on microfluidic platforms can minimize false-positive results, in addition to having potential for point-of-care applications. Here, we describe a polystyrene-toner (PS-T) centrifugal microfluidic device manually controlled by a fidget spinner for molecular diagnosis of COVID-19 by RT-LAMP, with integrated and automated colorimetric detection. The amplification was carried out in a microchamber with 5 µL capacity, and the reaction was thermally controlled with a thermoblock at 72 °C for 10 min. At the end of the incubation time, the detection of amplified RT-LAMP fragments was performed directly on the chip by automated visual detection. Our results demonstrate that it is possible to detect COVID-19 in reactions initiated with approximately 10-3 copies of SARS-CoV-2 RNA. Clinical samples were tested using our RT-LAMP protocol as well as by conventional RT-qPCR, demonstrating comparable performance to the CDC SARS-CoV-2 RT-qPCR assay. The methodology described in this study represents a simple, rapid, and accurate method for rapid molecular diagnostics of COVID-19 in a disposable microdevice, ideal for point-of-care testing (POCT) systems.

Mechanical/Physical Methods of Cell Disruption and Tissue Homogenization.

This chapter covers the various methods of mechanical cell disruption and tissue homogenization that are currently commercially available for processing small samples s < 1 mL) to larger multikilogram production quantities. These mechanical methods of lysing do not introduce chemicals or enzymes to the system. However, the energies required when using these "harsh," high mechanical energy methods can be enough to damage the very components being sought.The destruction of cell membranes and walls is effected by subjecting the cells (a) to shearing by liquid flow, (b) to exploding by pressure differences between inside and outside of cell, (c) to collision forces by impact of beads or paddles, or (d) a combination of these forces.Practical suggestions to optimize each method, where to acquire such equipment, and links to reference sources are included. Several novel technologies are presented.

Effects of rotor angle and time after centrifugation on the biological in vitro properties of platelet rich fibrin membranes.

This study evaluated the impact of rotor angle and time of storage after centrifugation on the in vitro biological properties of platelet-rich fibrin (PRF) membranes. Blood samples (n = 9) were processed with a vertical fixed-angle (V) or a swing-out horizontal (H) centrifuge, with 20-60 min of sample storage after centrifugation. Leukocytes, platelets, and red blood cells were counted, and fibrin architecture was observed by scanning electron microscopy (SEM). The release of FGF2, PDGFbb, VEGF, IL-6, and IL-1ß was measured after incubation on culture media for 7-21 days. Cell content was equivalent in all experimental groups (p > .05). The fibrin matrix was similar for fixed-angle and horizontal centrifugation. Horizontal centrifugation induced a twofold increase in PDGF and 1.7× increase on FGF release as compared to V samples, while IL-1ß was significantly reduced (p < .05). No significant difference was observed on the release of growth factors and cytokines at different times after centrifugation (p < .05). These data suggest that both angles of centrifugation produce PRF membranes with similar structure and cellularity, but horizontal centrifugation induces a higher release of growth factors. Higher times of storage after centrifugation did not impact on cell content and the release of growth factors.

Motion sickness symptoms during jumping exercise on a short-arm centrifuge.

Artificial gravity elicited through short-arm human centrifugation combined with physical exercise, such as jumping, is promising in maintaining health and performance during space travel. However, motion sickness symptoms could limit the tolerability of the approach. Therefore, we determined the feasibility and tolerability, particularly occurrence of motion sickness symptoms, during reactive jumping exercises on a short-arm centrifuge. In 15 healthy men, we assessed motion sickness induced by jumping exercises during short-arm centrifugation at constant +1Gz or randomized variable +0.5, +0.75, +1, +1.25 and +1.5 Gz along the body axis referenced to center of mass. Jumping in the upright position served as control intervention. Test sessions were conducted on separate days in a randomized and cross-over fashion. All participants tolerated jumping exercises against terrestrial gravity and on the short-arm centrifuge during 1 Gz or variable Gz at the center of mass without disabling motion sickness symptoms. While head movements markedly differed, motion sickness scores were only modestly increased with jumping on the short-arm centrifuge compared with vertical jumps. Our study demonstrates that repetitive jumping exercises are feasible and tolerable during short-arm centrifugation. Since jumping exercises maintain muscle and bone mass, our study enables further development of exercise countermeasures in artificial gravity.

Effects of gravity on flow rate estimations of a centrifugal blood pump using the eccentric position of a levitated impeller.

Implantable ventricular assist devices are a type of mechanical circulatory support for assisting the pumping of the heart. Accurate estimation of the flow rate through such devices is critical to ensure effective performance. A novel method for estimating the flow rate using the passively stabilized position of a magnetically levitated impeller was developed by our group. However, the performance of the method is affected by the gravity vector, which depends on the patient's posture. In this study, the effects of gravity on the flow estimation method are analyzed, and a compensation method is proposed. The magnetically levitated impeller is axially suspended and radially restricted by passive stability in a centrifugal blood pump developed by our group. The gravity effects were evaluated by analyzing the relationships between the radial position of the magnetically levitated impeller and the flow rate with respect to the gravity direction. Accurate estimation of the flow rate could not be achieved when the direction of gravity with respect to impeller was unknown. A mean absolute error of up to 4.89 L/min was obtained for flow rate measurement range of 0-5 L/min. However, analysis of the equilibrium of forces on the passively stabilized impeller indicated that the effects of gravity on the flow estimation could be compensated by performing additional measurements of the gravity direction with respect to impeller. The method for compensating the effects of gravity on the flow estimation should improve the performance of therapy with the implantable ventricular assist devices.

Impeller (straight blade) design variations and their influence on the performance of a centrifugal blood pump.

INTRODUCTION: The miniaturization of blood pumps has become a trend due to the advantage of easier transplantation, especially for pediatric patients. In small-scale pumps, it is much easier and more cost-efficient to manufacture the impeller with straight blades compared to spiral-profile blades. METHODS: Straight-blade impeller designs with different blade angles, blade numbers, and impeller flow passage positions are evaluated using the computational fluid dynamics method. Blade angles (θ = 0°, 20°, 30°, and 40°), blade numbers (N = 5, 6, 7, and 8), and three positions of impeller flow passage (referred to as top, middle, and bottom) are selected as the studied parametric values. RESULTS: The numerical results reveal that with increasing blade angle, the pressure head and the hydraulic efficiency increase, and the average scalar shear stress and the normalized index of hemolysis decrease. The minimum radial force and axial thrust are obtained when θ equals 20°. In addition, the minimum average scalar shear stress and normalized index of hemolysis values are obtained when N = 6, and the maximum values are obtained when N = 5. Regarding the impeller flow passage position, the axial thrust and the stagnation area forming in the impeller eye are reduced as the flow passage height declines. CONCLUSION: The consideration of a blade angle can greatly improve the performance of blood pumps, although the influence of the blade number is not very easily determined. The bottom position of the impeller flow passage is the best design.

Different Re-centrifugation Conditions for Infectious Disease Screening Using the Abbott I2000.

We investigated whether an ordinary centrifuge can achieve the standard centrifugal effect required according to specifications for infectious disease screening using the Abbott i2000. Samples were collected and centrifuged following a standard operating procedure (SOP). They were then divided into three groups according to the results of the initial screening tests: a negative group, weak-positive group, and positive group. Twenty negative samples and all weak-positive and positive samples were re-analyzed. Two tubes for each re-analyzed sample were centrifuged simultaneously, one for 10 min at 10 000 × g, per recommendations, and one for 10 min at 2750 × g. No significant difference was found between the groups using different centrifugal forces. There was a strong correlation in the quantitative values between the two conditions of centrifugation. Consistency analysis showed a Cronbach's alpha > 0.8 for detection of Treponema pallidum, human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B surface antigen in the three groups (negative group, weak-positive group, and positive group) under different centrifugation conditions. Strong consistency was found under different centrifugal conditions, regardless of the initial testing results. In conclusion, we conducted centrifugation steps in duplicate, according to infectious disease screening protocols. Our study showed that all samples should be centrifuged using a recommended relative centrifugal force after a proper clotting time, as in the standard operating procedure of our laboratory. In this way, we were able to obtain the same results using an ordinary centrifuge as those obtained using a high-speed centrifuge, such as the Abbott i2000.

Comparison of platelet-rich fibrin (PRF) produced using 3 commercially available centrifuges at both high (~ 700 g) and low (~ 200 g) relative centrifugation forces.

OBJECTIVES: Platelet-rich fibrin (PRF) has gained tremendous momentum in recent years as a natural autologous growth factor derived from blood capable of stimulating tissue regeneration. Owing to its widespread use, many companies have commercialized various centrifugation devices with various proposed protocols. The aim of the present study was to compare 3 different commercially available centrifuges at both high and low g-force protocols. MATERIALS AND METHODS: PRF was produced on three commercially available centrifuges including the IntraSpin Device (IntraLock), the Duo Quattro (Process for PRF), and Salvin (Salvin Dental). Two separate protocols were tested on each machine including the original leukocyte and platelet-rich fibrin (L-PRF) protocol (~ 700 RCF max (~ 400 RCF clot) for 12 min) as well as the advanced platelet-rich fibrin (A-PRF+) protocol (~ 200 g RCF max (~ 130 g RCF clot) for 8 min). Each of the tested groups was compared for cell numbers, growth factor release, scanning electron microscopy (SEM) for morphological differences, and clot size (both weight and length/width). RESULTS: The present study found that PRF clots produced utilizing the low-speed centrifugation speeds (~ 200 g for 8 min) produce clots that (1) contained a higher concentration of evenly distributed platelets, (2) secreted higher concentrations of growth factors over a 10 day period, and (3) were smaller in size. This was irrespective of the centrifugation device utilized and consistently observed on all 3 devices. The greatest impact was found between the protocols utilized (up to a 200%). Interestingly, it was further revealed that the centrifugation tubes used had a much greater impact on the final size outcome of PRF clots when compared to centrifugation devices. It was found that, in general, the Process for PRF tubes produced significantly greater-sized clots when compared to other commercially available tubes. The Salvin Dental tubes also produced significantly greater PRF clots when compared to the IntraLock tubes on each of the tested centrifugation devices. CONCLUSIONS: The present study demonstrated the reproducibility of a scientific concept (reduction in RCF produces PRF clots with more evenly distributed cells and growth factors) utilizing different devices. Furthermore, (and until now overlooked), it was revealed for the first time that the centrifugation tubes are central to the quality production of PRF. Future research investigating tube characteristics thus becomes critically important for the future optimization of PRF. CLINICAL RELEVANCE: This is the first study to reveal the marked impact of centrifugation tubes on the final production of PRF. Future study thus becomes markedly important to further optimize the quality of PRF-based matrices. It was further found that little variability existed between the centrifugation devices if optimized centrifugation protocols (lower centrifugation speeds) were utilized.

Low-cost multi-core inertial microfluidic centrifuge for high-throughput cell concentration.

We developed a low-cost multi-core inertial microfluidic centrifuge (IM-centrifuge) to achieve a continuous-flow cell/particle concentration at a throughput of up to 20 mL/min. To lower the cost of our IM-centrifuge, we clamped a disposable multilayer film-based inertial microfluidic (MFIM) chip with two reusable plastic housings. The key MFIM chip was fabricated in low-cost materials by stacking different polymer-film channel layers and double-sided tape. To increase processing throughput, multiplexing spiral inertial microfluidic channels were integrated within an all-in-one MFIM chip, and a novel sample distribution strategy was employed to equally distribute the sample into each channel layer. Then, we characterized the focusing performance in the MFIM chip over a wide flow-rate range. The experimental results showed that our IM-centrifuge was able to focus various-sized particles/cells to achieve volume reduction. The sample distribution strategy also effectively ensured identical focusing and concentration performances in different cores. Finally, our IM-centrifuge was successfully applied to concentrate microalgae cells with irregular shapes and highly polydisperse sizes. Thus, our IM-centrifuge holds the potential to be employed as a low-cost, high-throughput centrifuge for disposable use in low-resource settings.

Near-Physiological Cell Cycle Synchronization with Countercurrent Centrifugal Elutriation.

The bioreactor conditions and cell diversity in mammalian cell cultures are often regarded as homogeneous. Recently, the influence of various kinds of heterogeneities on production rates receives increasing attention. Besides spatial gradients within the cultivation system, the variation between cell populations and the progress of the cells through the cell cycle can affect the dynamics of the cultivation process. Strong metabolic up- and down-regulations leading to variable productivities, even in exponentially growing cell cultures, have been identified in CHO cell cultivations. Consequently, scientific studies of cell cycle-related effects and metabolic regulations require experiments utilizing cell cycle-enriched subpopulations. Importantly, the enrichment procedure itself must not strongly interfere with the cell culture under investigation. Such subpopulations can be generated by near-physiological countercurrent centrifugal elutriation, which is described in the following chapter. At first, a brief overview regarding the cell cycle, currently identified effects and commonly used methods, and their applicability is outlined. Then, the experimental setup and the synchronization itself are explained.

The use of a centrifuge-free RABiT-II system for high-throughput micronucleus analysis.

The cytokinesis-block micronucleus (CBMN) assay is considered to be the most suitable biodosimetry method for automation. Previously, we automated this assay on a commercial robotic biotech high-throughput system (RABiT-II) adopting both a traditional and an accelerated micronucleus protocol, using centrifugation steps for both lymphocyte harvesting and washing, after whole blood culturing. Here we describe further development of our accelerated CBMN assay protocol for use on high-throughput/high content screening (HTS/HCS) robotic systems without a centrifuge. This opens the way for implementation of the CBMN assay on a wider range of commercial automated HTS/HCS systems and thus increases the potential capacity for dose estimates following a mass-casualty radiological event.

Structure-dependent retention of steroid hormones by common laboratory materials.

The tendency of steroid molecules to adsorb to various materials, particularly plastics, has been known of for decades but has not received widespread attention in the scientific community, and a modern, systematic study is lacking. This adsorption is an important consideration for researchers working with steroid hormones as it could skew the results of various experiments. Here we show that steroids adsorb to various vessels used in experiments, including microcentrifuge tubes, glass vials, and cell culture plates, in a manner that depends on the steroid's molecular structure and on the type of vessel. The lipophilicity of steroids is a strong predictor of the degree of adsorption, with nearly 50 % of the most lipophilic steroid tested, pregnenolone, retained in a high-adsorbing microcentrifuge tube after one hour incubation of an aqueous pregnenolone solution followed by removal of the aqueous solvent. We also show the effects of other factors such as incubation time, centrifugation, and temperature on adsorption, and show that adsorption can be mostly prevented by the presence of serum proteins in steroid solutions and/or by the use of low-adsorbing tubes.

Dynamic Measurement Method for Bio-molecular Interactions by Using Centrifugal Force.

Measurement of single molecule interaction is performed by various micromanipulation techniques. However, a complex operation is required to perform the measurement, so the measurement throughput is low and a large amount of time is required for statistical analysis. In this paper, we propose a method for measuring intermolecular interactions that applies a tensile force to the intermolecular bond by using centrifugal force and can easily measure binding force. The binding force of an antigen-antibody reaction system was measured by the use of the proposed method as a proof of concept. The measured binding forces were distributed in the range of 19 - 133 pN. The values obtained by use of the proposed method were consistent with the conventional method. This result is promising for applications that require further development, such as the development of a biosensor as a novel measuring technique that is based on the intermolecular interaction measurement.