Standard Centrifugation Research Articles

Differential composition and yield of leukocytes isolated from various blood component leukoreduction filters

In Flanders, an estimated 300,000 leukoreduction filters are discarded as biological waste in the blood establishment each year. These filters are a possible source of fresh donor leukocytes for downstream purposes including research. We investigated leukocyte isolation from two types of filters either used for the preparation of platelet concentrates (PC-LRF) or erythrocyte concentrates (EC-LRF). Outcome parameters were leukocyte yield, differential count, turnaround time and effect of storage conditions. Leukocytes were harvested by reverse flow of a buffer solution. Control was the gold standard density gradient centrifugation of buffy coats. Total leukocyte number isolated from PC-LRF (1049 (± 40) x 106) was almost double that of control (632 (± 66) x 106) but the differential count was comparable. Total leukocyte number isolated from EC-LRF (78 (± 9) x 106) was significantly lower than control, but the sample was specifically enriched in granulocytes (81 ± 4%) compared to control (30 ± 1%). Isolation of leukocytes from either PC- or EC-LRF takes 20 min compared to 240 min for control density gradient centrifugation. Leukocyte viability is optimal when harvested on day 1 post donation (95 ± 0.9%) compared to day 3 (76.4 ± 2.4%). In conclusion, our study demonstrates that leukoreduction filters from specific blood component processing are easy to use and present a valuable source for viable leukocytes of all types.

Abstract 1121: Neutrophil Proteobacteria Sequences Increases After Open Aortic Surgery

Introduction: Bacterial extracellular vesicles (bEV) transport diverse cargoes, including nucleic acids, and play a crucial role during host-microbe interactions, including modifying host immune response. Neutrophil activation is prevalent in aortic disease pathogenesis, but the cause is unclear. The baseline neutrophilic bEV content and its dynamics are unknown and may provide insight into neutrophil activation in aortic disease. Research Question: Does open aortic surgery change neutrophil bEV content? Methods: Blood was collected pre and post-operatively from 6 patients undergoing open aortic surgery. Neutrophils were isolated using standard centrifugation techniques, and nucleic acids were purified. The V1-V3 16s rRNA segments were amplified with universal primers and sequenced using an Illumina MiSeq platform. Reads were processed in QIIME2 and aligned to reference banks of known bacterial sequences. Sequence abundance was analyzed with Kruskal-Wallis and Wilcoxon-rank tests. Results: The average patient age was 60.2 years, with 4 male patients and 4 current smokers. The most common phylum and genus represented in the neutrophil bacterial reads were Proteobacteria (Figure 1A) and Delftia , respectively. The relative abundance of Proteobacteria sequences increased overtime (p=0.042) from a median of 45.43% (IQR:35.3-52.0) to 70.8% (IQR:66.1-81.5, p=0.026) post-operatively, to 83.5% (IQR:60.7-89.7, p=0.004) on POD1 (Figure 1B). POD3 Proteobacteria sequence abundance was stable from POD1 at 81.4% (IQR:64.2-89.6). Conclusions: Neutrophils of patients with aortic disease rapidly change their bacterial sequence content in response to open aortic surgery. Bacteria in the phylum Proteobacteria change most dramatically and equilibrate between POD 1 and 3. Further studies will elucidate the impact changing bacterial sequence abundance and bEV content has on neutrophil activity and its relationship to disease pathogenesis.

OxyHbMeter-a novel bedside medical device for monitoring cell-free hemoglobin in the cerebrospinal fluid-proof of principle.

Delayed cerebral ischemia (DCI) occurs in up to one third of patients suffering from aneurysmal subarachnoid hemorrhage (aSAH). Untreated, it leads to secondary cerebral infarctions and is frequently associated with death or severe disability. After aneurysm rupture, erythrocytes in the subarachnoid space lyse and liberate free hemoglobin (Hb), a key driver for the development of DCI. Hemoglobin in the cerebrospinal fluid (CSF-Hb) can be analyzed through a two-step procedure of centrifugation to exclude intact erythrocytes and subsequent spectrophotometric quantification. This analysis can only be done in specialized laboratories but not at the bedside in the intensive care unit. This limits the number of tests done, increases the variability of the results and restricts accuracy. Bedside measurements of CSF-Hb as a biomarker with a point of care diagnostic test system would allow for a continuous monitoring for the risk of DCI in the individual patient. In this study, a microfluidic chip was explored that allows to continuously separate blood particles from CSF or plasma based on acoustophoresis. An in vitro test bench was developed to test in-line measurements with the developed microfluidic chip and a spectrometer. The proof of principle for a continuous particle separation device has been established with diluted blood and CSF samples from animals and aSAH patients, respectively. Processing 1 mL of blood in our microfluidic device was achieved within around 70 min demonstrating only minor deviations from the gold standard centrifugation (7% average error of patient samples), while saving several hours of processing time and additionally the reduction of deviations in the results due to manual labor.

Approaches for attaining clean bacterial fractions from complex environmental samples

Marine bacteria have been targeted by industry and pharmaceutics as genetic resources for highly active enzymes or novel lead compounds. Although numerous techniques have been introduced to isolate useful bacteria from the environment, we are still highly dependent on the conventional direct cultivation method to attain pure cultures. However, efficient bacterial isolation is hindered by several factors, including the presence of impurities. In this work, to demonstrate the significance of removing impurities and their impact on bacterial isolation, we employed two approaches: dielectrophoresis (DEP) and fluorescent D-amino acids (FDAA). We successfully attained clean bacterial fractions applicable for downstream processing using these approaches, uniquely designed to identify bacteria based on their characteristics and features. The diversity of bacteria attained by both approaches was investigated using 16S rRNA sequencing and compared to that attained by the standard differential centrifugation method. In addition, the viability of the isolates was also determined via direct cultivation. As a result, the separation of bacteria from impurities allowed for the identification of novel and useful bacteria unique to each approach. Successful cultivation also suggested that both approaches were applicable for attaining viable bacteria. In conclusion, removing impurities to attain clean bacterial fractions promotes the isolation of novel bacteria and thus could aid in the successful isolation of useful bacteria within complex environmental samples.

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.

Cell-Free Microbial DNA Analysis: Effects of Blood Plasma and Serum Quantity, Biobanking Protocols, and Isolation Kits.

Recent studies highlight the presence of bacterial sequences in the human blood, suggesting potential clinical significance for circulating microbial signatures. These sequences could presumably serve in the diagnosis, prediction, or monitoring of various health conditions. Ensuring the similarity of samples before bacterial analysis is crucial, especially when combining samples from different biobanks prepared under varying conditions (such as different DNA extraction kits, centrifugation conditions, blood collection tubes, etc.). In this study, we aimed to analyze the impact of different sample collection and nucleic acid extraction criteria (blood collection tube, centrifugation, input volume, and DNA extraction kit) on circulating bacterial composition. Blood samples from four healthy individuals were collected into three different sample collection tubes: K2EDTA plasma tube, sodium citrate plasma tube, and gel tube for blood serum. Tubes were centrifugated at standard and double centrifugation conditions. DNA extraction was performed using 100, 200, and 500 μL plasma/serum input volumes. DNA extraction was performed using three different isolation kits: Norgen plasma/serum cell-free circulating DNA purification micro kit, Applied Biosystems MagMAX cell-free DNA isolation kit, and Qiagen QIAamp MinElute cell-free circulating DNA mini kit. All samples were subjected to 16S rRNA V1-V2 library preparation and sequencing. In total, 216 DNA and 18 water control samples were included in the study. According to PERMANOVA, PCoA, Mann-Whitney, and FDR tests the effect of the DNA extraction kit on the microbiota composition was the greatest, whereas the type of blood collection tube, centrifugation type, and sample input volume for the extraction had minor effects. Samples extracted with the Norgen DNA extraction kit were enriched with Gram-negative bacteria, whereas samples extracted with the Qiagen and MagMAX kits were enriched with Gram-positive bacteria. Bacterial profiles of samples prepared with the Qiagen and MagMAX DNA extraction kits were more similar, whereas samples prepared with the Norgen DNA extraction kit were significantly different from other groups.

Transformed astrocytes confer temozolomide resistance on glioblastoma via delivering ALKBH7 to enhance APNG expression after educating by glioblastoma stem cells-derived exosomes.

Glioblastoma is the most malignant primary brain tumor in adults. Temozolomide (TMZ) stands for the first-line chemotherapeutic agent against glioblastoma. Nevertheless, the therapeutic efficacy of TMZ appears to be remarkably limited, because of low cytotoxic efficiency against glioblastoma. Besides, various mechanical studies and the corresponding strategies fail to enhancing TMZ curative effect in clinical practice. Our previous studies have disclosed remodeling of glial cells by GSCs, but the roles of these transformed cells on promoting TMZ resistance have never been explored. Exosomes were extracted from GSCs culture through standard centrifugation procedures, which can activate transformation of normal human astrocytes (NHAs) totumor-associated astrocytes (TAAs) for 3 days through detect the level of TGF-β, CD44 and tenascin-C. The secretive protein level of ALKBH7 of TAAs was determined by ELISA kit. The protein level of APNG and ALKBH7 of GBM cells were determined by Western blot. Cell-based assays of ALKBH7 and APNG triggered drug resistance were performed through flow cytometric assay, Western blotting and colony formation assay respectively. A xenograft tumor model was applied to investigate the function of ALKBH7 invivo. Finally, the effect of the ALKBH7/APNG signaling on TMZ resistance were evaluated by functional experiments. Exosomes derived from GSCs can activate transformation of normal human astrocytes (NHAs)to tumor-associated astrocytes (TAAs), as well as up-regulation of ALKBH7expression in TAAs. Besides, TAAs derived ALKBH7 can regulate APNG gene expression of GBM cells. After co-culturing with TAAs for 5 days, ALKBH7 and APNG expression in GBM cells were elevated. Furthermore, Knocking-down of APNG increased the inhibitory effect of TMZ on GBM cells survival. The present study illustrated a new mechanism of glioblastoma resistance to TMZ, which based on GSCs-exo educated TAAs delivering ALKBH7 to enhance APNG expression of GBM cells, which implied that targeting on ALKBH7/APNG regulation network may provide a new strategy of enhancing TMZ therapeutic effects against glioblastoma.

Mitochondrial function is abnormal in circulating blood monocytes of patients with chronic heart failure

Abstract Background Cardiac mitochondrial function is abnormal in heart failure (HF) and is likely to contribute to disease progression. We previously showed that in dogs with HF mitochondrial functional abnormalities exist in both failing cardiomyocytes and in circulating blood monocytes, and to similar extent. This finding suggested the possibility that mitochondrial function in circulating monocytes can serve as a surrogate for cardiac mitochondrial function. We also showed that mitochondrial abnormalities exist in cardiomyocytes of patients with chronic HF. Purpose In the present study we sought to test the difference between patients with advanced HF and age-matched controls in terms of circulating monocyte mitochondrial function. Methods We examined circulating blood monocytes from 10 patients evaluated in our advanced HF clinic and with left ventricular ejection fraction <35%. compared to 10 age-matched control persons without cardiac disease (CON). Peripheral blood samples (15 ml) were obtained from each participant and monocytes were isolated using standard Ficoll-Paque gradient centrifugation. Isolated monocytes were suspended in Agilent XF-DMEM base buffer for quantification of mitochondrial function with Seahorse XFe96 analyzer. The following key mitochondrial functional parameters were determined: basal respiration (BASEresp), ATP production-linked respiration (ATPresp), maximal respiration (MAXresp) and respiratory capacity (RC). Results BASEresp, ATPresp, MAXresp and RC were significantly lower in monocytes of patients with HF compared to NL subjects (BASEresp 2.7 ± 0.7 vs. 5.2 ± 1.6, p<0.05; ATPresp 10.0 ± 0.8 vs. 15.9 ± 1.0, p<0.05; MAXresp 36.5 ± 5.9 vs. 69.3 ± 3.8, p<0.05; RC 26.5 ± 5.5 vs. 53.5 ± 3.5, p<0.05). Conclusions Mitochondrial dysfunction is present in circulating blood monocytes of patients with chronic HF. These findings coupled to those in dogs with HF suggest the possibility that determination of mitochondrial function in peripheral monocytes may identify those patients with HF with cardiac energetic deficiencies. If validated this technique could help illuminate how cardiac energy deprivation relates to disease progression and potentially enable targeted therapies directed at mitochondria dysfunction.

Rapid, label-free enrichment of lymphocytes in a closed system using a flow-through microfluidic device.

The majority of adoptive cellular therapies are produced from peripheral mononuclear cells obtained via leukapheresis and further enriched for the cells of interest (e.g., T cells). Here, we present a first-of-its-kind closed system, which effectively removes ~85% of monocytes and ~88% of platelets, while recovering ~88% of concentrated T cells in a separate output stream, as the leukapheresis sample flows through a microfluidic device at 5 mL/min. The system is driven by a common peristaltic pump, enabled by a novel pressure wave dampener, and operates in a closed bag-to-bag configuration, without requiring any specialized, dedicated equipment. When compared to standard density gradient centrifugation on paired samples, the new system demonstrated a 1.5-fold increase in T cell recovery and a 2-fold reduction in inter-sample variability for this separation outcome. The T cell-to-monocyte ratio of the leukapheresis sample was increased to 20:1, whereas with density gradient processing it decreased to 2:1. As a result of superior purity and/or gentler processing, T cells enriched by the system showed a 2.7-times higher fold expansion during subsequent culture, and an overall 3.5-times higher cumulative yield. This centrifugation-free and label-free closed system for enriching lymphocytes could significantly simplify and standardize the manufacturing of life-saving cellular therapies.

Centriselect – A novel approach for centrifugal cell separation

Abstract The separation of bio-suspensions, such as blood, is of great interest for many biomedical applications, such as the extraction of leucocytes from whole blood, the laboratory analysis of specific cells or a mixed cell suspensions from bioreactors or the preparation of cellular products for cellbased therapy. In particular, mechanical separation processes have been established for the rapid separation of biosuspensions. The principle of mechanical separation is based on the specific density of the particles or the particle size. Thus, the corresponding processes are based on centrifugation and/or filtration. The standard process to prepare blood products from whole blood donations contains several steps. First the blood is separated into different phases by centrifugation and then further separated by filters containing specific filtration layers. The shortcomings of this process is the low yield due to bio-particles remaining in the filter and the high costs for equipment due to the use of multiple devices for centrifugation and filtration. Here we present the proof of principle of an adaptive, size-selective filter that addresses the above mentioned shortcomings. The filter is designed to combine the filtration and centrifugation process within one step. Therefore a novel filtration device was designed that can be used within a standard centrifugation tube. During centrifugation, the centrifugal force changes the filtration coefficient of the filter. This can be used to separate different sized of bio-particles at different centrifugation speeds. Moreover, the cells that were hold back in the filter can be removed afterwards by increasing the centrifugation speed. In this way, the filter is created to gradually release cells of a specific size yielding in a high purity of different cell fractions.

A Microfluidic Platform with an Embedded Miniaturized Electrochemical Sensor for On-Chip Plasma Extraction Followed by In Situ High-Sensitivity C-Reactive Protein (hs-CRP) Detection.

Blood testing is a clinical diagnostic tool to evaluate physiological conditions, the immune system response, or the presence of infection from whole blood samples. Although conventional blood testing can provide rich biological information, it usually requires complicated and tedious whole blood processing steps operated by benchtop instruments and well-experienced technicians, limiting its usage in point-of-care (POC) settings. To address the above problems, we propose a microfluidic platform for on-chip plasma extraction directly from whole blood and in situ biomarker detection. Herein, we chose C-reactive protein (CRP) as the target biomarker, which can be used to predict fatal cardiovascular disease (CVD) events such as heart attacks and strokes. To achieve a rapid, undiluted, and high-purity on-chip plasma extraction, we combined two whole blood processing methods: (1) anti-D immunoglobulin-assisted sedimentation, and (2) membrane filtration. To perform in situ CRP detection, we fabricated a three-dimensional (3D) microchannel with an embedded electrochemical (EC) sensor, which has a modular design to attach the blood collector and buffer reservoir with standard Luer connectors. As a proof of concept, we first confirmed that the dual plasma extraction design achieved the same purity level as the standard centrifugation method with smaller sample (100 µL of plasma extracted from 400 µL of whole blood) and time (7 min) requirements. Next, we validated the functionalization protocol of the EC sensor, followed by evaluating the detection of CRP spiked in plasma and whole blood. Our microfluidic platform performed on-chip plasma extraction directly from whole blood and in situ CRP detection at a 0.1-10 μg/mL concentration range, covering the CVD risk evaluation level of the high-sensitivity CRP (hs-CRP) test. Based on the above features, we believe that this platform constitutes a flexible way to integrate the processing of complex samples with accurate biomarker detection in a sample-to-answer POC platform, which can be applied in CVD risk monitoring under critical clinical situations.

Effect of centrifugation force and time on the analysis of lactate dehydrogenase and potassium in the serum samples

Introduction and Aim: Any imperfection that occurs during any stage of the testing process is described as laboratory error. Increasing requirements of biochemical tests, numerous patient samples and automation has forced laboratory work to be carried out at a faster speed. Few studies are shown to investigate the influence of settings of centrifugation of less than 10 minutes on the laboratory result in serum. Thus, our study was aimed to see the effect of centrifugation force and time on the analysis of lactate dehydrogenase(LDH) and potassium from serum samples. Methodology: Samples were collected from 61 healthy volunteers. 5ml was taken in two separate BD vacutainer serum tubes. Tube 1 was centrifuged for 2000g for 10 minutes, tube 2 for 5 minutes 3000g, and analysed for LDH and potassium. Results: A significant difference was observed between 5 min (U/L) (3000g) and 10 min (U/L) (2000g) with LDH and 5 min (mmol/l) 3000g and 10 min (mmol/l) 2000g with potassium. Conclusion: LDH and potassium levels were found to be raised by increasing the centrifugal force to 3000g. Hence, the standard centrifugation protocol of 10 min at 2000 or 2500 rpm is to be followed to get the accurate results.

THE THREE MUSKETEERS: A RETROSPECTIVE COHORT STUDY EXAMINING EMBRYO QUALITY IN IVF-ICSI CYCLES USING VARYING SPERM SELECTION METHODS

The limited literature comparing testicular vs. ejaculated sperm for assisted reproduction technology (ART) is rife with heterogenous results. While a meta-analysis found that testicular sperm was superior to ejaculated sperm with respect to embryo quality and pregnancy rates, a well-designed recent study concluded that intracytoplasmic sperm injection (ICSI) outcomes with the use of Testicular Sperm Extraction (TESE) samples compared with ejaculated sperm were equivalent (1). Moreover, studies comparing Zymot™, a microfluidic sperm separation device vs standard density gradient centrifugation (DGC) remain conflicting (2).

Growth Factor Release within Liquid and Solid PRF.

Aim: The purpose of this study was to obtain data concerning growth factor release within liquid and solid platelet-rich fibrin (PRF) matrices and to estimate the amount of potential interindividual variations as a basis for further preclinical and clinical trials. Therefore, we aimed to determine possible differences in the release of growth factors between liquid and solid PRF. Materials and Methods: Blood samples obtained from four subjects were processed to both liquid and solid PRF matrices using a standard centrifugation protocol. Five growth factors (vascular endothelial growth factor, VEGF; epidermal growth factor, EGF; platelet-derived growth factor-BB, PDGF-BB; transforming growth factor-β1, TGF-β1; and matrix metallopeptidase 9, MMP-9) have been evaluated at six time points by ELISA over a total observation period of 10 days (1 h, 7 h, 1 d, 2 d, 7 d, and 10 d). Results: Growth factor release could be measured in all samples at each time point. Comparing liquid and solid PRF matrices, no significant differences were detected (p > 0.05). The mean release of VEGF, TGFβ-1, PDGF-BB, and MMP-9 raised to a peak at time point five (day 7) in both liquid and solid PRF matrices. VEGF release was lower in liquid PRF than in solid PRF, whereas those of PDGF-BB and MMP-9 were higher in liquid PRF than in solid PRF at all time points. EGF had its peak release already at time point two after 7 h in liquid and solid matrices (hour 7 EGF solid: mean = 180 pg/mL, SD = 81; EGF liquid: mean = 218 pg/mL, SD = 64), declined rapidly until day 2, and had a second slight peak on day 7 in both groups (day 7 EGF solid: mean = 182 pg/mL, SD = 189; EGF liquid: mean = 81 pg/mL, SD = 70). Conclusions: This study detected growth factor release within liquid and solid PRF matrices with little variations. Further preclinical trials are needed to precisely analyze the growth factor release in larger samples and to better understand their effects on wound healing in different clinical indications.

Autoverification-based algorithms to detect preanalytical errors: Two examples

The preanalytical phase of testing accounts for the majority of the errors. Software-based quality rules, such as autoverification, can assist in preanalytical error detection; therefore, preventing erroneous results from being reported. Two autoverification rules, turbidity/lipemia, and pseudohypoglycemia/pseudohyperkalemia alarms, are highlighted. Increased sample turbidity may arise from several causes outside of lipemia. Typically, this can be resolved by clarifying the sample with standard centrifugation. Truly lipemic specimens typically require higher centrifugation speeds and greater centrifugation time. At our facility, 96% of direct bilirubin (DBIL), 95% of aspartate transaminase (AST), and 98% of alanine transaminase (ALT) turbidity/lipemia alarms were found to be from sample turbidity versus lipemia. Secondly, a pseudohypoglycemia/pseudohyperkalemia rule was employed for specimens with delayed separation from cellular material. Of the total potassium results >6.0 mmol/L or glucose results <40 mg/dL (2.2 mmol/L), 30% and 50% respectively were noted to have delayed sample separation.

Abstract 2212: Racially disparate serum levels of inflammatory cytokines, satiety and stress hormones, and exosomal microRNAs in women with or without a breast cancer diagnosis

Abstract Breast cancer (BC) is the most common non-cutaneous malignancy and the second leading cause of cancer-related death in American women. Health disparities in incidence and clinical outcome are also reported among different racial groups, most notably African American (AA) women, who are often diagnosed at a young age with aggressive BC and exhibit greater mortality than Caucasian American (CA) women. Since socioeconomic difficulties can have tremendous impact on psychophysiology besides limiting the access to optimal healthcare, we examined the serum levels of stress (cortisol) and satiety (leptin) hormones as well as inflammatory cytokines (resistin and interleukin-6/IL-6) in AA and CA women. To observe a potential epigenomic connection, we also performed profiling of a targeted set of exosomal microRNAs in serum samples. The study was conducted under an Institutional Review Board (IRB) approved protocol. All subjects participated voluntarily, and their consents were obtained. Serum levels of resistin, IL-6, leptin, and cortisol were quantified by Enzyme linked Immunosorbent Assay (ELISA) using commercial kits. Exosomes were isolated using precipitation method and recovered by standard centrifugation. Total RNA was isolated from exosomal preps and subjected to stem-loop RT-PCR for quantitation of a set of inflammation-associated microRNAs. We found that the levels of resistin, leptin, IL-6 and cortisol were higher in women with a BC diagnosis than non-BC subjects. Moreover, AA women with or without BC showed significantly higher levels of these cytokines and hormones in their serum as compared to the CA women with or without a BC diagnosis, respectively. We also observed differential expression of several microRNAs in serum of BC women as compared to their normal counterparts, of which five (miR511, miR33a, miR27a, miR6794, miR143-3p) exhibited highest presence in serum exosomes of AA women with BC. Together, these findings suggest that relatively greater exposure of minority women to social stressors may have epigenomic consequences and may potentially be linked to the observed BC racial health disparities. Citation Format: Sarabjeet Kour Sudan, Kunwar Somesh Vikramdeo, Amod Sharma, Sachin Kumar Deshmukh, Sanjeev Kumar Srivastava, Teja Poosarla, Nicolette P. Holliday, Donna L. Dyess, Ajay P. Singh, Seema Singh. Racially disparate serum levels of inflammatory cytokines, satiety and stress hormones, and exosomal microRNAs in women with or without a breast cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2212.

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Microfluidic preparation of spermatozoa for ICSI produces similar embryo quality to density-gradient centrifugation: a pragmatic, randomized controlled trial.

Does processing of spermatozoa for IVF with ICSI by a microfluidic sperm separation device improve embryo quality compared with density-gradient centrifugation? Patients randomized to microfluidic sperm preparation had similar cleavage- and blastocyst-stage embryo quality and clinical and ongoing pregnancy rates to those who underwent standard sperm processing for IVF with ICSI. Microfluidic sperm preparation can isolate spermatozoa for clinical use with minimal DNA fragmentation but with unclear impact on clinical outcomes. A prospective randomized controlled trial of 386 patients planning IVF from June 2017 through September 2021 was carried out. One hundred and ninety-two patients were allocated to sperm processing with a microfluidic sperm separation device for ICSI, while 194 patients were allocated to clinical standard density-gradient centrifugation (control) at an academic medical centre. In an intention to treat analysis, there were no differences in high-quality cleavage-stage embryo fraction [66.0 (25.8)% control versus 68.0 (30.3) microfluidic sperm preparation, P = 0.541, absolute difference -2.0, 95% CI (-8.5, 4.5)], or high-quality blastocyst fraction [37.4 (25.4) control versus 37.4 (26.2) microfluidic sperm preparation, P = 0.985, absolute difference -0.6 95% CI (-6, 5.9)] between groups. There were no differences in the clinical pregnancy or ongoing pregnancy rates between groups. The population studied was inclusive and did not attempt to isolate male factor infertility cases or patients with a history of elevated sperm DNA fragmentation. Microfluidic sperm separation performs similarly to density-gradient centrifugation in sperm preparation for IVF in an unselected population. No external funding to declare. M.P.R. is a member of the Clinical Advisory Board for ZyMōt® Fertility, Inc. NCT03085433. 21 March 2017. 16 June 2017.

Microfluidic Paper-Based Blood Plasma Separation Device as a Potential Tool for Timely Detection of Protein Biomarkers.

A current challenge regarding microfluidic paper-based analytical devices (µPAD) for blood plasma separation (BPS) and electrochemical immunodetection of protein biomarkers is how to achieve a µPAD that yields enough plasma to retain the biomarker for affinity biosensing in a functionalized electrode system. This paper describes the development of a BPS µPAD to detect and quantify the S100B biomarker from peripheral whole blood. The device uses NaCl functionalized VF2 filter paper as a sample collection pad, an MF1 filter paper for plasma retention, and an optimized microfluidic channel geometry. An inverted light microscope, scanning electron microscope (SEM), and image processing software were used for visualizing BPS efficiency. A design of experiments (DOE) assessed the device’s efficacy using an S100B ELISA Kit to measure clinically relevant S100B concentrations in plasma. The BPS device obtained 50 μL of plasma from 300 μL of whole blood after 3.5 min. The statistical correlation of S100B concentrations obtained using plasma from standard centrifugation and the BPS device was 0.98. The BPS device provides a simple manufacturing protocol, short fabrication time, and is capable of S100B detection using ELISA, making one step towards the integration of technologies aimed at low-cost POC testing of clinically relevant biomarkers.

Influence of Single Layer Centrifugation with Canicoll on Semen Freezability in Dogs.

Simple SummaryFreezing dog semen is not always possible due to low quality sperm or poor survival during freezing. In order to make this assisted reproductive technique available to a larger number of dogs, this study investigated the benefit of selecting the best spermatozoa before freezing using single layer centrifugation (SLC). The results indicated that this technique was effective in separating spermatozoa according to their quality, although this resulted in losing some good quality spermatozoa. After thawing, spermatozoa centrifuged by SLC were of better quality than after standard centrifugation. However, spermatozoa from suboptimal quality semen did not survive freezing as well as spermatozoa from semen of optimal quality, even after SLC. Single layer centrifugation, therefore, makes it possible to obtain better quality spermatozoa after thawing but is not sufficient on its own to improve the inferior freezing ability of spermatozoa from suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile.This study evaluated how semen selection by single layer centrifugation (SLC) with Canicoll affects semen freezability in dogs. A total of eighteen ejaculates, collected from dogs with optimal and suboptimal semen quality (optimal: normal morphology (NM) ≥ 80%, n = 9; suboptimal: NM between 60 and 79%, n = 9), were divided into two aliquots and subjected to standard centrifugation or SLC before cryopreservation. Motility, NM, membrane integrity, mitochondrial membrane potential (MMP), and DNA integrity were improved in fresh samples after SLC, regardless of semen quality, but at the expense of some good quality spermatozoa. After thawing, NM and membrane integrity were improved in SLC-selected semen in both semen qualities. Interestingly, MMP was also higher but only in optimal quality semen. Still, spermatozoa from suboptimal quality semen did not survive freezing to the same extent as spermatozoa from optimal quality semen, even after selecting superior spermatozoa. Semen selection with Canicoll is, therefore, an effective technique to isolate a subpopulation of high-quality spermatozoa and obtain sperm samples of better quality after thawing, but is not sufficient to improve the intrinsic inferior freezability of suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile.