Effect of storage and single layer centrifugation before cryopreservation on stallion sperm cryosurvival.

The objectives of this study were to evaluate the effect of a short, cooled storage before cryopreservation on sperm progressive motility (PM) and compare the effect of different centrifugation methods on post-thaw PM of stored samples. Semen was diluted in chilling extender and aliquoted in 6 protocols: i) Standard centrifugation (SC) followed by freezing; ii) Single Layer Centrifugation (SLC) followed by freezing; iii) Storage for 8 h/5 °C before SC; iv) Storage for 8 h/5 °C before SLC; v) Storage for 8 h/15 °C before SC; and vi) Storage for 8 h/15 °C before SLC. PM was assessed before centrifugation, after centrifugation, and post-thawing. Stallions were classified as "good freezers" (GF) or "bad freezers" (BF). The PM in samples immediately frozen was greater than in the stored ones (71.98 ± 14.2, 52.91 ± 17.8, 53.93 ± 18.9 for no storage, 5 ºC storage and 15 ºC storage, respectively) (P˂ 0.0001). There was an effect of storage condition (p ˂ 0.0001), centrifugation method (p ˂ 0.0001), and freezability (P=0.0016), with an interaction between them (P= 0.0004), on PM after centrifugation. Post-thaw PM was greater in samples treated by SLC than in samples processed by SC, for all storage conditions (p ˂ 0.05). All BF stallions 'showed post-thaw PM ˂ 30 % when samples were previously stored. Storage at 5 ºC or 15º C for 8 h maintains an appropriate quality in GF stallions. Applying a sperm selection technique as SLC is suggested to improve post-thaw motility, allowing GF straws to be frozen after storage, although BF semen should be prepared by SLC immediately after collection.

Delayed centrifugation weakens the in vitro biological properties of platelet-rich fibrin membranes.

OBJECTIVE: To investigate how delayed blood centrifugation affects the composition of the resultant platelet rich fibrin membrane (PRF, a concentrated growth factor preparation) and its biological effects towards gingival fibroblasts. MATERIALS AND METHODS: Blood samples were collected from 18 healthy individuals and centrifuged immediately (T-0), or after a 1-6-minute delay (T-1-6, respectively), to generate PRF. Each PRF membrane was weighed. T-0 and T-6 membranes were incubated for 48 h in cell culture medium at 37 °C to create PRF "releasates" (soluble factors released from the PRF). Human gingival fibroblasts were incubated for 48 h with or without the releasates, followed by RNA isolation and real-time polymerase chain reaction to measure expression of select genes associated with granulation tissue formation, angiogenesis and wound contraction. Additional T-0 and T-6 membranes were used for visualization of leucocyte nuclei and platelets by immunostaining. RESULTS: Immediate centrifugation (T-0) resulted in the largest membranes, T-6 membranes being on average 29% smaller. Leucocytes and platelets were significantly more abundant in T-0 than in T-6 samples. Majority of the fibroblast genes studied were consistently either upregulated or downregulated by the T-0 PRF releasates. However, centrifugation after a 6-minute delay significantly weakened the fibroblast responses. CONCLUSIONS: Delayed centrifugation resulted in smaller PRF membranes with fewer leucocytes and platelets and also significantly reduced on the expression of a set of healing-related gingival fibroblast genes. CLINICAL RELEVANCE: The higher expression of wound healing-related genes in gingival fibroblasts by the immediately-centrifuged PRF membranes may increase their biological properties in clinical use.

[Study of centrifuge conditions for preparing rabbit leukocyte-poor platelet-rich plasma by single centrifugation].

Objective: To explore the best centrifuge condition for preparing rabbit leukocyte-poor platelet-rich plasma (LP-PRP) by using single centrifugation method. Methods: Sixteen healthy New Zealand rabbits, aged 3-4 months, were utilized in the investigation. A total of 15 mL anticoagulated blood was extracted from the central ear artery of each rabbit, with a repeat of the blood collection procedure after 1 and 2 months. The obtained blood specimens were individually subjected to centrifugation at a radius of 16.7 cm and speeds of 1 200, 1 300, 1 400, and 1 500 r/min (equivalent to centrifugal forces of 269× g, 315× g, 365× g, and 420× g) for durations of 2, 3, 4, and 5 minutes, resulting in a total of 16 groups. Following centrifugation, collect plasma from each group to a distance of 1.5 mL from the separation plane. The volumes, platelet enrichment coefficient, and platelet recovery rates of LP-PRP in each group, under varying centrifugation conditions, were methodically computed and subsequently compared. Results: The volume of LP-PRP obtained under all centrifugation conditions ranged from 1.8 to 7.6 mL. At a consistent centrifugal speed, an extension of centrifugation time leaded to a significant increase in the volume of LP-PRP, accompanied by a declining trend in the platelet enrichment coefficient of LP-PRP. When centrifuged for 2 minutes, the volume of LP-PRP at speeds of 1 200 and 1 300 r/min was less than 2.0 mL, while the volume of LP-PRP obtained under other conditions was more than 2.0 mL. When centrifuged for 4 and 5 minutes, the volume of LP-PRP obtained at each speed was more than 4 mL. LP-PRP with a platelet enrichment coefficient more than 2.0 could be prepared by centrifuging at 1 200 r/min for each time group and 1 300 r/min for 2 and 3 minutes, and the highest LP-PRP platelet enrichment coefficient could be obtained by centrifugation for 2 minutes at a speed of 1 200 r/min. The platelet recovery rates of LP-PRP obtained by centrifugation at 1 200 r/min for 4 and 5 minutes, as well as centrifugation at 1 400 r/min for 5 minutes, were both greater than 60%. There was no significant difference between the groups when centrifuged at 1 200 r/min for 4 and 5 minutes ( P>0.05). Conclusion: In the process of preparing rabbit LP-PRP using a single centrifugation method, collecting 15 mL of blood and centrifuging at a radius of 16.7 cm and speed of 1 200 r/min for 4 minutes can prepare LP-PRP with a volume exceeding 2.0 mL, platelet enrichment coefficient exceeding 2.0, and platelet recovery rate exceeding 60%. This centrifugal condition can achieve the optimal LP-PRP action parameters in the shortest possible time.

Reduced bacterial load in stallion semen by modified single layer centrifugation or sperm washing.

The presence of bacteria poses a significant challenge to the quality of stallion semen used in artificial insemination. The bacterial content of insemination doses arises from various sources, such as the healthy stallion, environment, and collection equipment, and is implicated in fertility problems as well as reduced sperm quality during storage. The conventional approach of adding antibiotics to semen extenders raises concerns about antimicrobial resistance and potential negative effects on sperm characteristics, and may not be effective in inhibiting all bacteria. The objective of this study was to determine whether an innovative alternative to antibiotic usage - centrifugation through a single layer of a low density colloid (SLC) - could reduce the bacterial load in stallion semen, and to compare sperm characteristics in samples arising from this procedure, or simple extension of the ejaculate in semen extender, or from sperm washing, i.e. adding extender and then centrifuging the sample to allow the removal of most of the seminal plasma and extender. Eighteen semen samples were collected from six stallions. The semen samples were split and extended prior to washing or SLC, or received no further treatment other than extension. After preparation aliquots from each type of sample were sent for bacteriological examination; the remaining samples were stored for up to 72 h, with daily checks on sperm quality. The low density colloid SLC outperformed sperm washing or extension for bacterial reduction, effectively removing several bacterial species. The bacterial load in the samples was as follows: extended semen, 16 ± 6.7 × 105; washed, 5.8 ± 2.0 × 105; SLC, 2.3 ± 0.88 × 105, p < 0.0001. In addition, SLC completely removed some bacterial species, such as Staphylococcus xylosus. Although there is no selection for robust spermatozoa with the low density colloid, sperm motility, membrane integrity, and DNA fragmentation were not different to washed sperm samples. These findings suggest that SLC with a low density colloid offers a promising method for reducing bacterial contamination in stallion semen without resorting to antibiotics.

Comparison of two processing techniques on the sperm quality of semen contaminated with urine.

Urospermia in stallions can occur intermittently, consistently, or as an isolated event, and may result in reduced sperm quality which is often assumed to reduce fertility. Although sperm quality declines in urospermic ejaculates, fertility has not been assessed in mares bred with urine contaminated semen. The aims of this study were to compare sperm quality after simple dilution (SD), cushioned centrifugation (CC) alone, or cushioned centrifugation combined with a 40 % silane-coated silica solution (SC) in semen contaminated with 0, 20, or 40 % (v/v) urine. Sperm quality values tended to decrease as the percent urine increased within all treatments (SD, CC, SC) after 24 h of cooled storage. However, SC treated groups had higher sperm quality compared to SD and CC when exposed to 20 or 40 % (v/v) urine. Differences in pregnancy rates among treatment groups (SD with 0 or 40 % (v/v) urine, or 40 % (v/v) urine followed by SC) were unable to be detected.

Cushioned and high-speed centrifugation improve sperm recovery rate but affect the quality of fresh and cryopreserved feline spermatozoa.

The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires a small volume of the insemination dose with a high concentration, not easily reached with the actual frozen technique in this species. Centrifugation is widely used to concentrate spermatozoa for several purposes, but this process is detrimental to spermatozoa. This study verified the effects of conventional and cushioned centrifugation on fresh and cryopreserved feline spermatozoa. To this, semen was collected from 20 toms, grouped in seven pools and diluted. After dilution, the pools were divided into two aliquots, the first used for centrifugation on fresh semen, and the second, after freezing, on cryopreserved semen. Centrifugation regimens were: conventional at 500×g, conventional at 1000×g, and cushioned (iodixanol) at 1000×g. The sperm recovery rate was calculated for the three centrifugation regimens, and sperm kinematics, membrane and acrosome integrity, and plasma membrane stability on viable spermatozoa were assessed as endpoints. The data reported in this study showed that the centrifugation at 500×g resulted in negligible effects on both fresh and cryopreserved spermatozoa, but the lower recovery rate (62.4 ± 3.1 % and 60.2 ± 1.6 %, respectively) underlines the loss of a large proportion of spermatozoa, unfavourable in a species with small total sperm ejaculated. On the other hand, the centrifugation at 1000×g improved the recovery rate (86.9 ± 4.3 % and 89.8 ± 2.4 % in fresh and cryopreserved samples, respectively), but was more deleterious for feline spermatozoa, especially in cryopreserved samples (i.e. total motility of 40.7 ± 5.4 % compared with 57.2 ± 9.8 % in cryopreserved uncentrifuged samples, P < 0.05), resulting in artificial insemination doses of lower quality. The recovery rate in cushioned centrifugation appeared less efficient, likely due to the small volume of feline samples, which makes difficult the separation of sperm pellet and cushioned fluid. Interestingly, in cryopreserved samples centrifuged at 1000×g the number of viable spermatozoa with membrane destabilization (31.3 ± 3.2 %) was greater than uncentrifuged (4.1 ± 0.7 %, P < 0.05) and those centrifuged at 500×g (9.8 ± 1.3 %, P < 0.05), suggesting modifications induced by the cryopreservation amplifies centrifugation sublethal damage on feline spermatozoa. Cushioned centrifugation on cryopreserved samples showed kinematics similar to uncentrifuged samples, but higher viable spermatozoa with membrane destabilization (37.4 ± 3.4 % vs 4.1 ± 0.7 %; P < 0.05). In felines, g-force is crucial for sperm recovery rate during centrifugation, with better results at 1000×g; on the other hand, greater g-forces could have a significant impact on the quality of feline insemination dose, especially in cryopreserved samples.

Fully Automated Multiple Standard Addition on a Centrifugal Microfluidic System.

We herein describe a novel centrifugal microfluidic system to achieve multiple standard additions, which could minimize the effects of matrix interference and consequently lead to more accurate and reliable measurements of analyte concentrations in complex samples. The system leverages laser-irradiated ferrowax microvalves to automatically control fluid transfer on the disc without the need for external pumps or pressure systems, simplifying the procedures and eliminating the need for manual intervention. The disc incorporates metering chambers with rationally designed varying sizes, which could lead to the formation of six standard addition samples very rapidly in just 2.5 min. The final solutions are designed to contain a target component at gradually increasing concentrations but have an equal final volume containing the same amount of an analyte solution, thereby equalizing the matrix effect that is supposedly caused by the unknown components in the analyte solution. By utilizing this design principle, we were able to successfully quantify a model target component, salivary thiocyanate ions, that could be used as a biomarker for exposure to tobacco smoke. Our centrifugal microfluidic system holds great promise as a powerful analytical tool to achieve fully automated diagnostic microsystems involving a standard addition process.

Pre-freezing selection of Holstein bull semen with the BoviPure colloid as double- or single-layer centrifugation improves the post-thawing quality.

Artificial insemination (AI) is critical for breeding in the dairy industry. High-merit bulls can present low freezability, hampering genetic dissemination. Thawed semen can be improved using density gradient centrifugation (DGC) with colloids, but little information deals with the pre-freezing application. Thus, the BoviPure colloid (optimized for bull spermatozoa) was tested for pre-freezing application as the usual double-layer (DLC) versus single-layer (SLC, quick and economical). Semen from twelve Holstein-Friesian bulls was extended with OPTIXcell extender, frozen (Control), or processed by SLC or DLC and frozen. Sperm were assessed pre-freezing for motility and viability and post-thawing (directly and after 4 h 38 °C) for apoptosis, capacitation status, acrosomal damage, mitochondrial activity, cytoplasmic and mitochondrial reactive oxygen species (ROS), and chromatin status (SCSA for DNA fragmentation and chromatin compaction and monobromobimane, mBBr, for disulfide bridges evaluation). The DGC improved parameters post-thawing (e.g., 57.5%±10.1 motility vs. control 53.3% ± 11.2) at the cost of sperm loss (sperm recovery of DGC 14.4% ± 2.5 and SLC 17.4% ± 2.5). DNA fragmentation (%DFI) decreased (0.21% ± 0.53 vs. control 1.30% ± 0.10), and SLC reduced chromatin compaction. A clustering procedure separated lesser (LF) and greater freezability (GF) bulls. LF samples were especially benefited by DGC, with SLC providing better post-thawing results for this group. In conclusion, pre-freezing DGC improved sperm parameters post-thawing, potentially improving the cryopreservation of low-freezability semen from high-merit bulls. SLC, quicker and economical, would be preferable since it showed similar or higher performance than DLC.

Investigation of three common centrifugation protocols for platelet-rich fibrin (PRF) as a bio-carrier for ampicillin/sulbactam: a prospective trial.

OBJECTIVES: Different platelet-rich fibrin (PRF) protocols exist and are known to differ in resulting mechanical and bioactive properties. Centrifugation parameters may also influence drug release, in particular antibiotics, when using PRF as a bio-carrier. We thus evaluated three common protocols regarding effects on the bio-carrier properties. MATERIALS AND METHODS: In a prospective trial comprising 33 patients, we compared different protocols for PRF as a bio-carrier for ampicillin/sulbactam (SAM). Blood samples were taken shortly after a single dose of ampicillin/sulbactam (2 g/1 g) was administered to patients intravenously. PRF was obtained by centrifugation and three protocols were used: protocol A (1300 rpm, 8 min, RCF-max = 208 g), B (2300 rpm, 12 min, RCF-max = 652 g), and C (1500 rpm, 14 min, RCF-max = 276 g). The antibacterial activity of PRF was investigated against five oral species in vitro, based on agar diffusion methodology. RESULTS: The study demonstrates that a single dose of SAM is sufficient to reach high concentrations in PRF in all protocols (150 µg/ml), which is comparable to the plasma SAM concentration. Antibacterial activity was inferred from the diameter of inhibition zones seen in agar diffusion tests using PRF discs. Protocol B resulted in the largest inhibition zones. One-way ANOVA revealed statistically improved results for protocol B for some bacteria. CONCLUSIONS: The study provides valuable data on PRF antibiotic enrichment, notably SAM. A single dose of SAM is sufficient to reach clinically relevant concentrations in PRF. CLINICAL RELEVANCE: These findings potentially extend the application of PRF, for example in patients with osteonecrosis of the jaw or in oral surgery (e.g., stick bone).

Evaluation of a single-use disk stack centrifuge for improved efficiency and sustainability at 1000 L GMP manufacturing scale.

Direct depth filtration is an established technology for single-use harvest operation. Advantages of direct depth filtration include familiarity with depth filtration in downstream processes and simplicity of the operation. Drawbacks include low capacity, large footprint, labor-intensive set-up, high water use, and high waste in the form of discarded filters. Single-use centrifugation is emerging as an alternative to depth filtration for the single-use harvest step. Within the single-use centrifugation space, disc stack centrifugation represents the newest entrant. In this study, we evaluated the performance of the GEA kytero single-use disc stack centrifuge to clarify two monoclonal antibody-producing cell culture fluids. The separation performance of the GEA kytero centrifuge varied between the two cell culture fluids, with differences in centrate turbidity and centrate filterability measured. A comparison was then performed to determine resource savings, compared to direct two-stage depth filtration, when using a GEA kytero centrifuge to harvest a 1000 L bioreactor. The analysis concluded that replacement of the first stage of depth filters with a GEA kytero centrifuge has the potential to decrease the required second stage depth filtration area by up to 80%. The decrease in depth filter area resulting from the use of the GEA kytero would result in a decrease in the harvest step footprint, a decrease in buffer volume required to prime and rinse depth filters, and a decrease in the volume of plastic waste. An economic comparison of the GEA kytero single-use centrifuge against a direct depth filtration step found that for a 1000 L harvest step, the GEA kytero centrifuge may reduce costs by up to 20% compared with two-stage direct depth filtration.

Glass wool column filtration for stallion sperm selection: A comparative analysis with the single-layer colloid centrifugation.

Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

Effects of platelet-rich fibrin produced by three centrifugation protocols on bone neoformation in defects created in rat calvaria.

This study evaluated the potential of Leukocyte-platelet-rich fibrin (L-PRF; fixed angle centrifugation protocol), Advanced-platelet-rich fibrin (A-PRF; low-speed fixed angle centrifugation protocol), and Horizontal-platelet-rich fibrin (H-PRF; horizontal centrifugation protocol) in bone neoformation in critical size defects (CSDs) in rat calvaria. Thirty-two rats were divided into groups: Control (C), L-PRF, A-PRF, and H-PRF. 5 mm diameter CSDs were created in the animals' calvaria. Defects from group Control (C) were filled with blood clots, while defects from groups L-PRF, A-PRF, and H-PRF were filled with respective platelet-rich fibrin (PRF) membranes. L-PRF, A-PRF, and H-PRF were prepared from animal blood collection and specific centrifugation protocols. At 14 and 30 days, calcein (CA) and alizarin (AL) injections were performed, respectively. Animals were euthanized at 35 days. Microtomographic, laser confocal microscopy, and histomorphometric analyzes were performed. Data were statistically analyzed (ANOVA, Tukey, p < .05). L-PRF, A-PRF, and H-PRF groups showed higher values of bone volume (BV), newly formed bone area (NFBA), and precipitation of CA and AL than the C group (p < .05). The H-PRF group showed higher values of BV, number of trabeculae (Tb. N), NFBA, and higher precipitation of AL than the A-PRF and L-PRF groups (p < .05). Therefore, it can be concluded that: i) L-PRF, A-PRF, and H-PRF potentiate bone neoformation in CSDs in rat calvaria; ii) H-PRF demonstrated more biological potential for bone healing.

After tooth loss, the alveolar bone (which supports the teeth) undergoes a natural process called bone remodeling, which can lead to significant decreases in bone height and thickness over time. Faced with the need to replace missing teeth, especially when it comes to dental implants, the lack of supporting tissues can compromise their correct positioning, leading to negative impacts on the success and longevity of the treatment. Therefore, over the years, several materials and procedures have been proposed to preserve and regenerate oral tissues. Leukocyte-platelet-rich fibrin (L-PRF) consists of a membrane obtained by centrifuging the patient's blood in a fixed-angle centrifuge, allowing cells to be available to stimulate tissue regeneration directly at the place of action. Several reports demonstrate high potential in stimulating the formation of new tissues using L-PRF. In recent years, new protocols have been proposed to increase cell concentration and improve the regenerative potential of these membranes, changing the speed and time of centrifugation and introducing horizontal centrifugation. However, there still needs to be concrete evidence of the superiority of the new protocols in relation to the original protocol. In this study, we evaluated the healing of defects created in rat calvaria using platelet aggregates obtained through different centrifugation protocols. Within the limits of this study, it can be concluded that platelet aggregates improve bone healing, and horizontal centrifugation promotes more satisfactory results compared to fixed-angle protocols.

Recent advances in microfluidic cell separation to enable centrifugation-free, low extracorporeal volume leukapheresis in pediatric patients.

Leukapheresis is a common extracorporeal procedure for leukodepletion and cellular collection. During the procedure, a patient's blood is passed through an apheresis machine to separate white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), which are then returned to the patient. Although it is well-tolerated by adults and older children, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of a typical leukapheresis circuit represents a particularly large fraction of their total blood volume. The reliance of existing apheresis technology on centrifugation for separating blood cells limits the degree to which the circuit ECV could be miniaturized. The rapidly advancing field of microfluidic cell separation holds excellent promise for devices with competitive separation performance and void volumes that are orders of magnitude smaller than their centrifugation-based counterparts. This review discusses recent advancements in the field, focusing on passive separation methods that could potentially be adapted to perform leukapheresis. We first outline the performance requirements that any separation method must meet to replace centrifugation-based methods successfully. We then provide an overview of the passive separation methods that can remove WBCs from whole blood, focusing on the technological advancements made in the last decade. We describe and compare standard performance metrics, including blood dilution requirements, WBC separation efficiency, RBC and PLT loss, and processing throughput, and discuss the potential of each separation method for future use as a high-throughput microfluidic leukapheresis platform. Finally, we outline the primary common challenges that must still be overcome for these novel microfluidic technologies to enable centrifugation-free, low-ECV leukapheresis in the pediatric setting.

Automation in the Thrombosis and Hemostasis Laboratory.

Automation continues to advance into hemostasis and thrombosis laboratories. Integration of hemostasis testing into an existing chemistry track systems and adoption of a separate hemostasis track systems are important considerations. Unique issues must be addressed to maintain quality and efficiency when automation is introduced. Among other challenges, this chapter discusses centrifugation protocols, incorporation of specimen-check modules in the workflow, and inclusion of tests amenable to automation.

Low-density colloid centrifugation removes bacteria from boar semen doses after spiking with selected species.

Single-layer centrifugation (SLC) with a low-density colloid is an efficient method for removing contaminating microorganisms from boar semen while recovering most spermatozoa from the original sample. This study tested the performance of this technique, using 50-ml tubes, by spiking commercial semen doses prepared without antibiotics with selected bacterial species followed by storage at 17 °C. The doses were spiked up to 102/ml CFU (colony forming units) of the bacteria Burkholderia ambifaria, Pseudomonas aeruginosa, and Staphylococcus simulans. The semen was processed by SLC (15 ml of sample and 15 ml of colloid) with the colloid Porcicoll at 20% (P20) and 30% (P30), with a spiked control (CTL) and an unspiked control (CTL0), analyzing microbiology and sperm quality on days 0, 3 and 7. SLC completely removed B. ambifaria and S. simulans, considerably reducing P. aeruginosa and overall contamination (especially P30, ∼104 CFU/ml of total contamination on day 7, median). Sperm viability was lower in P20 and P30 samples at day 0, with higher cytoplasmic ROS. Still, results were similar in all groups on day 3 and reversed on day 7, indicating a protective effect of SLC (possibly directly by removal of damaged sperm and indirectly because of lower bacterial contamination). Sperm chromatin was affected by the treatment (lower DNA fragmentation and chromatin decondensation) and storage (higher overall condensation on day 7 as per chromomycin A3 and monobromobimane staining). In conclusion, SLC with low-density colloids can remove most bacteria in a controlled contamination design while potentially improving sperm quality and long-term storage at practical temperatures.

Preanalytical temperature and storage stability of specific IgE antibodies in serum.

Allergen-specific serum immunoglobulin E (sIgE) levels are a cornerstone in allergy diagnostics. While immunoglobulins are known to be relatively stable molecules, the influence of preanalytical factors on the stability of sIgE is not thoroughly investigated. We studied the effect of several preanalytical factors: (1) delayed centrifugation of serum samples in tubes with separating gel for 10, 24 and 48 h, (2) prolonged storage at 5 °C for 3, 7, 10 and 14 days, (3) storage tube type (primary tube with separating gel or secondary tube), (4) repeated freeze-thawing cycles, and (5) prolonged storage at -20 °C for 4 and 8 weeks. We found that sIgE is stable at room temperature for 48 h before centrifugation and for 10 days at 5 °C after centrifugation. There was no effect of the separating gel after storing serum for 1 week in the freezer. However, storage for 4-8 weeks, and introducing more than one freeze-thaw cycle resulted in a larger variation of sIgE levels. In conclusion, we found that sIgEs are stable under various preanalytical conditions, which allows for flexible handling of samples for a comprehensive portfolio of sIgE analyses.

Integrating Bio-Sensing Array with Blood Plasma Separation on a Centrifugal Platform.

Numerous immunoassays have been successfully integrated on disc-based centrifugal platforms (CDs) over the last 20 years. These CD devices can be used as portable point-of-care (POC) platforms with sample-to-answer capabilities where bodily fluids such as whole blood can be used as samples directly without pre-processing. In order to use whole blood as a sample on CDs, centrifugation is used to separate red blood cells from plasma on CDs. There are several techniques for using specific fluidic patterns in the centrifugal fluidic network, such as reciprocation, that enhances the sensitivity of the immunoassays, including those using microarray antigen membranes. Present work demonstrates, for the first time, simultaneous integration of blood plasma separation (BPS) and reciprocation on the CD platform. The integrated design allows plasma that is separated from the red blood cells in a sedimentation chamber to flow into the reciprocation chamber via a narrow connecting channel of 0.5 mm × 0.5 mm cross-section. Due to the small cross-section of the connecting channel, there is no inflow of the red blood cell into the reciprocation chamber during subsequent fluidic operations of the CD. While no inflow of the red blood cells into the reciprocation chamber was observed, the conditions of 20 g jerk acceleration were also simulated in ANSYS finite element analysis software, and it was found that the CD design that was used is capable of retaining red blood cells in the sedimentation chamber. Experimentally, the isolation of red blood cells in the sedimentation chamber was confirmed using the ImageJ image processor to detect the visible color-based separation of the plasma from the blood. A fluorescent analyte testing on the bio-sensing array of the presented novel integrated design and on the standard reciprocation design CD was conducted for 7 min of reciprocation in each case. The test analyte was Europium Streptavidin Polystyrene analyte (10-3 mg/mL) and the microarray consisted of Biotin bovine serum albumin (BSA) dots. The fluorescent signals for the standard and integrated designs were nearly identical (within the margin of error) for the first several minutes of reciprocation, but the fluorescent signal for the integrated design was significantly higher when the reciprocation time was increased to 7 min.

Resolving Molecular Heterogeneity with Single-Molecule Centrifugation.

For many classes of biomolecules, population-level heterogeneity is an essential aspect of biological function─from antibodies produced by the immune system to post-translationally modified proteins that regulate cellular processes. However, heterogeneity is difficult to fully characterize for multiple reasons: (i) single-molecule approaches are needed to avoid information lost by ensemble-level averaging, (ii) sufficient statistics must be gathered on both a per-molecule and per-population level, and (iii) a suitable analysis framework is required to make sense of a potentially limited number of intrinsically noisy measurements. Here, we introduce an approach that overcomes these difficulties by combining three techniques: a DNA nanoswitch construct to repeatedly interrogate the same molecule, a benchtop centrifuge force microscope (CFM) to obtain thousands of statistics in a highly parallel manner, and a Bayesian nonparametric (BNP) inference method to resolve separate subpopulations with distinct kinetics. We apply this approach to characterize commercially available antibodies and find that polyclonal antibody from rabbit serum is well-modeled by a mixture of three subpopulations. Our results show how combining a spatially and temporally multiplexed nanoswitch-CFM assay with BNP analysis can help resolve complex biomolecular interactions in heterogeneous samples.

Effect of relative centrifugal force on the biological properties of liquid platelet-rich fibrin produced via horizontal centrifugation.

OBJECTIVES: Platelet-rich fibrin (PRF) in liquid form has shown advantages in tissue engineering including acting as injectable fillers and drug carriers. However, few studies have investigated the best relative centrifugal force (RCF) for preparing liquid PRF. The aim of the present study was to find out optimal centrifugation force for preparing liquid PRF. MATERIALS AND METHODS: Liquid PRF was prepared using horizontal centrifugation (liquid H-PRF) with RCF ranging from 100 g, 300 g, 500 g, to 700 g for 8 min. The volume, weight, solidification time, and tensile properties were subsequently investigated. Scanning electron microscopy (SEM) and rheologic tests were carried out to investigate the microstructure and rheologic properties of liquid H-PRF after natural polymerization. The total number, concentration, and distribution of cells within each liquid H-PRF was evaluated by complete blood count (CBC) analysis and hematoxylin-eosin staining. RESULTS: As RCF values increased, the volume and weight of liquid H-PRF both increased accordingly. SEM images revealed that as the centrifugal force increased, the fibrin bundles became thinner with a denser fibrin network, and rheologic tests revealed improved mechanical properties. CBC analysis demonstrated that 500 g group had the highest number of leukocytes and neutrophils, whereas 100 g group yielded the highest concentration of leukocytes and platelets. Furthermore, histological analysis suggests that cells obtained by 500 g for 8 min were most evenly distributed in liquid H-PRF. CONCLUSIONS: In summary, the present study provided insights into the contents of liquid H-PRF prepared at different centrifugation forces, enabling clinicians to choose proper centrifugation forces based on their needs. CLINICAL RELEVANCE: The present findings provide theoretical basis for clinical choice of liquid H-PRF protocol from mechanical, cell contents, and histological aspects.

A new technique for separating platelet-rich plasma by a copolymer device － without a centrifugation process.

BACKGROUND: Platelet-rich plasma (PRP) is beneficial for clinical applications in various medical fields. Although commercial PRP preparation kits are already available in the market, most of these kits employ centrifugation. METHODS: We used a new cationic copolymer coating on a polyurethane (PU) sponge to promote platelet separation from the blood. This copolymer showed no cytotoxicity against cell viability or hemolysis. We further evaluated the efficiency of the new PRP preparation device by comparing it with that of a commercially available kit (RegenKit-THT). RESULTS: We demonstrated that PRP obtained using copolymer device contains high concentrations of platelets and angiogenic growth factors (epidermal growth factor, vascular endothelial growth factor-A, growth differentiation factor 2, and interleukin-8). The separated PRP also displayed beneficial effects on cell migration, angiogenesis, and matrix metalloproteinase gene expression. CONCLUSION: Based on these results, we developed a cationic copolymer-coated PU sponge as a PRP preparation device without the need for any centrifugation.