Rapid Centrifugation Research Articles

Quantification of neurofilament light and glial fibrillary acidic protein in finger-prick blood.

An accurate diagnosis of neurodegenerative disease and traumatic brain injury is important for prognostication and treatment. Neurofilament light and glial fibrillary acidic protein (GFAP) are leading biomarkers for neurodegeneration and glial activation that are detectable in blood. Yet, current recommendations require rapid centrifugation and ultra-low temperature storage post-venepuncture. Here, we investigated if these markers can be accurately measured in finger-prick blood using dried plasma spot cards. Fifty patients (46 with dementia; 4 with traumatic brain injury) and 19 healthy volunteers underwent finger-prick and venous sampling using dried plasma spot cards and aligned plasma sampling. Neurofilament light and GFAP were quantified using a Single molecule array assay and correlations between plasma and dried plasma spot cards assessed. Biomarker concentrations in plasma and finger-prick dried plasma spot samples were significantly positively correlated (neurofilament light ρ = 0.57; GFAP ρ = 0.58, P < 0.001). Finger-prick neurofilament light and GFAP were significantly elevated after acute traumatic brain injury with non-significant group-level increases in dementia (91% having Alzheimer's disease dementia). In conclusion, we present preliminary evidence that quantifying GFAP and neurofilament light using finger-prick blood collection is viable, with samples stored at room temperature using dried plasma spot cards. This has potential to expand and promote equitable testing access, including in settings where trained personnel are unavailable to perform venepuncture.

866. A new protocol for bacterial identification and carbapenemases by MALDI-TOF and lateral flow assay directly from positive blood culture: MALDI-FAST

Abstract Background Infectious diseases are a global public health problem, accounting for more than 20% of the mortality burden. Klebsiella pneumoniae and Pseudomonas aeruginosa caused more than 1.3 million deaths globally in 2019. Antimicrobial resistance (AMR) is a major factor in this mortality. Rapid molecular test for the detection of the blaKPC gene improves treatment time and mortality in patients with bacteremia caused by carbapenem-resistant Enterobacterales, however, the use of molecular biology techniques may not be optimal in multiple resourced care settings limited. Our goal is to perform rapid identification of Gram-negative bacilli and detection of carbapenemases directly from blood cultures using the combination of mass spectrometry (MALDI-TOF) and the lateral flow immunochromatography test.4 Methods We analyzed 97 bottles of positive blood cultures detected by the BACT/Alert 3D system (BioMérieux®). Our new method (MALDI-FAST) uses 0.5ml of blood obtained from the positive blood culture bottle for Gram-negative bacilli, from which a pellet is obtained by rapid centrifugation in 3 minutes. This material is processed both for bacterial identification by MALDI-TOF mass spectrophotometry (Bruker®), and rapid detection of carbapenemases by NG Test Carba 5 lateral flow immunochromatography (NG Biotech®). The results obtained were compared with the gold standard RT-PCR FilmArray Panel BCID 2.0 (BioMérieux®) Results Of the 97 blood culture bottles, 94/97 (96.9%) were correctly identified to the species level and 3/97 (3.1%) were polymicrobial blood cultures. Klebsiella pneumoniae was detected in 39 samples, E. coli in 24, and 34 other microorganisms including non-fermenting bacteria. Excellent concordance (kappa index 1,000) was achieved in 31/97 blood cultures (31.9%) that presented carbapenemases, 18/31 KPC (18.6%) and 13/31 with KPC and NDM coproduction (13.4%). 66/97(68.15%) bottles did not present carbapenemase. Conclusion Our protocol presents very good results for bacterial identification by mass spectrometry and carbapenemase detection by lateral flow immunoassay in a few minutes directly from the positive blood culture, becoming a fast and cost-effective tool to contribute to patient management. Disclosures All Authors: No reported disclosures

Abstract 16947: Sotagliflozin, a Dual SGLT 1 and 2 Inhibitor, Has High Membrane Affinity and Hydrocarbon Core Location Compared With Empagliflozin

Background: The dual SGLT-1 and 2 inhibitor sotagliflozin (sota) reduced atherothrombotic events and hospitalization for worsening heart failure in patients with diabetes in clinical trials (SCORED, SOLOIST). Unlike selective SGLT2 inhibitors such as empagliflozin (empa), sota binds with higher affinity to SGLT1, which is located in the intestine, kidney, heart, brain, and vascular endothelial cells. To elucidate the physico-chemical basis for differences in tissue distribution and receptor affinity, we compared the effects of these drugs on membrane binding and molecular location. Methods: Membranes reconstituted from cardiac phospholipid and cholesterol were used to measure drug partitioning across a range of cholesterol concentrations. Multilamellar vesicles were prepared as mixtures of cardiac phospholipid (CPL), cholesterol, and each agent at 30 μM or vehicle. Drug-membrane partitioning was determined by rapid centrifugation and UV/Vis spectrophotometry. In parallel experiments, we used small angle x-ray scattering (SAXS) to identify drug location in model membranes containing phosphatidylcholine and cholesterol. Results: Sota had a membrane partition coefficient (8590 ± 2078) that was 16-fold higher than empa (519 ± 195, p&lt;0.001) in CPL membranes. This high membrane affinity was preserved over a range of cholesterol levels compared to empa (p&lt;0.001). Consistent with high membrane affinity, SAXS analysis showed that sota had a location evidenced by a broad increase in electron density in the hydrocarbon core extending to approximately 20 Å from the membrane center. By contrast, empa had a location limited to the polar headgroup region, consistent with its lower lipophilicity and tissue penetration. Conclusions: Sota had high membrane affinity and distinct location in the hydrocarbon core compared to empa. This may contribute to the effect of its dual SGLT-1 and 2 inhibition in vivo across various tissues including the heart. These distinct membrane interactions may also partially explain the reduced atherothrombotic risk as demonstrated in outcome trials with sota but not selective SGLT-2 inhibitors.

Influence of reduced centrifugation time on clinical chemistry analytes and literature review

Abstract Objectives Centrifugation is a time-consuming step which increases the turnaround time (TAT) in laboratories. A few studies have addressed the effect of altering centrifugation settings on analytical quality for clinical chemistry analytes, and most of these studies have used collection tubes with gel separators. However, gel separator tubes may be unsuitable for some laboratories because they are slightly more expensive than tubes without gel separators and are not appropriate for some special tests. The aim of this study was to investigate the effect of centrifugation conditions on clinical chemistry analytes. Methods We compared centrifugation times of 7 min at 2,200×g and 5 min at 2,750×g with the manufacturer’s protocol of 10 min at 1,300×g as the reference condition. Twenty general chemistry analytes were studied in lithium heparin plasma tubes without gel separators. Results For all analytes except carbon dioxide (CO2), no significant differences in analyte results were observed when the centrifugation time was reduced. Deming regression and Bland–Altman plots demonstrated an acceptable clinical concordance within the limits of total allowable error for all analytes between the two rapid centrifugation conditions with the reference centrifugation condition. Conclusions Our results confirmed that alternate centrifugation conditions for either 7 min at 2,200×g or 5 min at 2,750×g of samples collected in lithium heparin tubes without gel are acceptable for clinical chemistry analytes. Our data support using centrifugation at higher speeds for shorter times to improve TAT without altering the quality of the analytical results.

Generation of Red Blood Cell Nanovesicles as a Delivery Tool.

Extracellular vesicles (EVs) are natural membranous vesicles with immense potential as drug delivery tools. However, their large-scale production remains a huge technical challenge, is time consuming, and expensive. Thus, EV mimetics (nanovesicles) generated from easily sourced red blood cells (RBCs) have gained vested interest as an effective and scalable drug delivery system. Their surface proteins (e.g., CD47) inherited from parental RBCs also improve their biocompatibility and bioavailability. Here, we outline a step-by-step guide for large-scale production of RBC nanovesicles using one-step extrusion method coupled to rapid density-cushion centrifugation. We also outline protocol for their extensive biophysical characterization (size and morphology using single particle analysis and cryogenic electron microscopy), and in-depth mass spectrometry-based proteome characterization. Finally, we outline two strategies (active loading during extrusion vs. passive loading via diffusion) to incorporate pharmacological compound(s) into nanovesicles and detect their loading using spectrophotometry.

Impact of rapid centrifugation on routine coagulation assays in South Africa.

The recommendation for coagulation blood samples is to centrifuge at 4000 revolutions per minute (rpm) for 15 min to produce platelet-poor plasma before analysis. Rapid centrifugation, defined as centrifuging samples at higher speeds for shorter durations, could potentially reduce turn-around times (TAT), provided sample integrity is maintained. This study assessed the impact of rapid centrifugation on routine coagulation assay results. Blood samples were collected from volunteers at Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, from September to November 2021. Samples were centrifuged using Method A, the current standard (4000 rpm/15 min), Method B (4000 rpm/10 min), Method C (5000 rpm/10 min) and Method D (5000 rpm/5 min). Platelet count, prothrombin time, activated partial thromboplastin time, thrombin time (TT), fibrinogen and D-dimer levels were analysed and results from Methods B, C and D compared to reference Method A. Platelet-poor plasma was obtained from all samples (n = 60) using Methods A and B, and from 33/60 (55%) samples using Methods C and D. Differences between Method A and Methods C and D for normal prothrombin time, normal D-dimer and abnormal TT results were statistically significant. Prothrombin time results correlated strongly across all methods, while TT and D-dimer results correlated poorly. Activated partial thromboplastin time and fibrinogen results showed no significant differences across all methods. Rapid centrifugation at 4000 rpm/10 min (Method B) showed results consistent with the reference method. This method could potentially reduce the overall TAT for routine coagulation assays.

FcMBL magnetic bead-based MALDI-TOF MS rapidly identifies paediatric blood stream infections from positive blood cultures.

Rapid identification of potentially life-threatening blood stream infections (BSI) improves clinical outcomes, yet conventional blood culture (BC) identification methods require ~24-72 hours of liquid culture, plus 24-48 hours to generate single colonies on solid media suitable for identification by mass spectrometry (MS). Newer rapid centrifugation techniques, such as the Bruker MBT-Sepsityper® IVD, replace culturing on solid media and expedite the diagnosis of BCs but frequently demonstrate reduced sensitivity for identifying clinically significant Gram-positive bacterial or fungal infections. This study introduces a protocol that utilises the broad-range binding properties of an engineered version of mannose-binding lectin linked to the Fc portion of immunoglobulin (FcMBL) to capture and enrich pathogens combined with matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) MS for enhanced infection identification in BCs. The FcMBL method identified 94.1% (64 of 68) of clinical BCs processed, with a high sensitivity for both Gram-negative and Gram-positive bacteria (94.7 and 93.2%, respectively). The FcMBL method identified more patient positive BCs than the Sepsityper® (25 of 25 vs 17 of 25), notably with 100% (3/3) sensitivity for clinical candidemia, compared to only 33% (1/3) for the Sepsityper®. Additionally, during inoculation experiments, the FcMBL method demonstrated a greater sensitivity, identifying 100% (24/24) of candida to genus level and 9/24 (37.5%) top species level compared to 70.8% (17/24) to genus and 6/24 to species (25%) using the Sepsityper®. This study demonstrates that capture and enrichment of samples using magnetic FcMBL-conjugated beads is superior to rapid centrifugation methods for identification of BCs by MALDI-TOF MS. Deploying the FcMBL method therefore offers potential clinical benefits in sensitivity and reduced turnaround times for BC diagnosis compared to the standard Sepsityper® kit, especially for fungal diagnosis.

Oral glucose tolerance test to diagnose gestational diabetes mellitus: Impact of variations in specimen handling

To improve birth outcomes, all pregnant women without known diabetes are recommended for an oral glucose tolerance test (OGTT) to screen for hyperglycaemia in pregnancy (diabetes in pregnancy or gestational diabetes mellitus (GDM)). This narrative review presents contemporary approaches to minimise preanalytical glycolysis in OGTT samples with a focus on GDM diagnosis using criteria derived from the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study. The challenges of implementing each approach across a diverse Australian healthcare setting were explored. Many Australian sites currently collect and transport OGTT samples at ambient temperature in sodium fluoride (NaF) tubes which is likely to lead to missed diagnosis of GDM in a significant proportion of cases. Alternative preanalytical solutions should be pragmatic and tailored to individual settings and as close as possible to the preanalytical conditions of the HAPO study for correct interpretation of OGTT results. Rapid centrifugation of barrier tubes to separate plasma could be suitable in urban settings provided time to centrifugation is strictly controlled. Tubes containing NaF and citrate could be useful for remote or resource poor settings with long delays to analysis but the impact on the interpretation of OGTT results should be carefully considered. Testing venous blood glucose at the point-of-care bypasses the need for glycolytic inhibition but requires careful selection of devices with robust analytical performance. Studies to evaluate the potential error of each solution compared to the HAPO protocol are required to assess the magnitude of misdiagnosis and inform clinicians regarding the potential impact on patient safety and healthcare costs.

Parallel Reaction Monitoring Mode for Atenolol Quantification in Dried Plasma Spots by Liquid Chromatography Coupled with High-Resolution Mass Spectrometry

In this study, we reported a rapid, sensitive, robust, and validated method for atenolol quantification in dried plasma spots (DPS) by liquid chromatography with high-resolution mass spectrometry (LC-HRMS) using parallel reaction monitoring mode (PRM). Aliquots of 25 µL human plasma were placed onto Whatman 903 Cards and air-dried. Disks (3.2 mm internal diameter) were punched, and a 100 µL working internal standard solution was added to each sample and then incubated on a shaker for 15 min at 40 °C, followed by rapid centrifugation (10,000× g, 10 s). The supernatant was transferred into 300 µL vials for subsequent LC–HRMS analysis. After chromatographic separation, atenolol and the internal standard were quantified in positive-ion parallel reaction monitoring mode by detection of all target product ions at 10 ppm tolerances. The total time of the analysis was 5 min. The calibration curve was linear in the range of 5–1000 ng/mL with interday and intraday precision levels and biases of &lt;14.4%, and recovery was 62.9–81.0%. The atenolol in DPS was stable for ≥30 days at 25 and 4 °C. This fully validated method is selective and suitable for atenolol quantitation in DPS using LC–HRMS.

Hemolysis and blood gas analysis: it’s time for a change!

Modern blood gas analyzers are not able to identify hemolysis, lipemia and icterus; therefore, the aim of this study was to assess the influence of hemolysis on blood gas samples. Blood gas analysis represents an essential part in the diagnosis and treatment of critically ill patients, including those affected by the pandemic coronavirus disease 2019 (COVID-19). Hemolysis, lipemia, and icterus, are causes of clinical misinterpretation of laboratory tests. A total of 1244 blood gas specimens were collected over a one-week period from different clinical wards, including the Emergency Department, and were assessed for serum indices on Cobas C6000 CE (Roche Diagnostics, Mannheim, Germany). The prevalence of hemolysis, lipemia, and icterus were 5%, 12%, and 14%, respectively. Sample storage at room temperature, delivery to central laboratory using pneumatic tube system, as well as small sample size, strongly affected blood gas parameters (p < .01). Hemolysis led to an increase in analytical bias for pH, pO2, and potassium, and a significant decrease for pCO2, HCO3 −, sodium, and Ca2+ (p <.01). Currently, hemolysis detection systems are not yet widespread, and a rapid centrifugation of samples after blood gas analysis along with the assessment of serum indices represent the only prompt approach to identify unsuitable results, avoiding pitfalls in clinical decision-making, although it cannot be applied to the Emergency Department routine. Blood gas analyzers manufacturers and suppliers should implement automated built-in serum indices detection systems.

Fatty Acid Determination in Human Milk Using Attenuated Total Reflection Infrared Spectroscopy and Solvent-Free Lipid Separation.

This study introduces the first mid-infrared (IR)-based method for determining the fatty acid composition of human milk. A representative milk lipid fraction was obtained by applying a rapid and solvent-free two-step centrifugation method. Attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy was applied to record absorbance spectra of pure milk fat. The obtained spectra were compared to whole human milk transmission spectra, revealing the significantly higher degree of fatty acid-related spectral features in ATR FT-IR spectra. Partial least squares (PLS)-based multivariate regression equations were established by relating ATR FT-IR spectra to fatty acid reference concentrations, obtained with gas chromatography-mass spectrometry (GC-MS). Good predictions were achieved for the most important fatty acid sum parameters: saturated fatty acids (SAT, R2CV = 0.94), monounsaturated fatty acids (MONO, R2CV = 0.85), polyunsaturated fatty acids (PUFA, R2CV = 0.87), unsaturated fatty acids (UNSAT, R2CV = 0.91), short-chain fatty acids (SCFA, R2CV = 0.79), medium-chain fatty acids (MCFA, R2CV = 0.97), and long-chain fatty acids (LCFA, R2CV = 0.88). The PLS selectivity ratio (SR) was calculated in order to optimize and verify each individual calibration model. All mid-IR regions with high SR could be assigned to absorbances from fatty acids, indicating high validity of the obtained models.

Effect of Rapid Centrifugation Using High Centrifugal Force on Procoagulant Activity in Plasma Samples.

International guidelines for plasma preparation are not always observed because high-speed centrifugation reduces turnaround times. This study assessed the effect of rapid centrifugation on sample quality and clotting time. Blood samples were obtained from five healthy volunteers. Normal blood samples were spiked with unfractionated heparin to produce abnormal samples. The normal and abnormal coagulation ability samples were centrifuged at 1,500 x g for 15 minutes, 2,000 x g for 10 minutes (both according to international guidelines), or 3,500 x g for 7 minutes (rapid centrifugation). Microparticle procoagulant activity (MP activity), prothrombin time (PT), and activated partial thromboplastin time (APTT) were measured in the supernatant plasma. Rapid centrifugation caused a significant increase in MP activity compared to the two recommended conditions and significantly shortened clotting times, particularly APTT in the abnormal samples. Rapid centrifugation should not be used for routine processing of blood samples for coagulopathy screening and monitoring patients on anticoagulant therapy.

Workflow Mapping-A Q-Probes Study of Preanalytic Testing Processes: A College of American Pathologists Q-Probes Study of 35 Clinical Laboratories.

Workflow mapping is a tool used to characterize operational processes throughout most industries and to identify non-value-added activities. To develop a set of workflow mapping tools to compare the sequence and timing of activities, including waiting steps, used by clinical laboratories to process specimens during the preanalytic testing phase. Laboratories enrolled in this College of American Pathologists Q-Probes study created workflow maps detailing the steps they used to process specimens from the time of sample arrival in the laboratory to the time of sample delivery to chemistry analyzers. Enrollees recorded the sequence and types of steps involved in specimen processing and the time needed to complete each step. Institution average total specimen processing times (SPTs) and the number of steps required to prepare samples varied widely among institutions. Waiting steps, that is, steps requiring specimens to wait before advancing to the next process step, and specimen centrifugation consumed the greatest amount of processing times for both routine and STAT testing. Routine and STAT testing SPTs were shorter at institutions that used rapid centrifuges to prepare samples. Specimen processes requiring more sample waiting steps and computer entry steps had longer aggregate total process times than those with fewer such steps. Aggregate specimen processing times may be shortened by reducing the number of steps involving sample waiting and computer entry activities. Rapid centrifugation is likely to reduce overall average institutional SPTs.

Trypanosome infections in naturally infected horses and donkeys of three active sleeping sickness foci in the south of Chad

BackgroundEquine trypanosomiases are complex infectious diseases with overlapping clinical signs defined by their mode of transmission. Despite their economic impacts, these diseases have been neglected by the scientific community, the veterinary authorities and regulatory organizations. To fill the observed knowledge gap, we undertook the identification of different trypanosome species and subspecies naturally infecting horses and donkeys within the Chadian sleeping sickness focus. The objective of the study was to investigate the potential role of these domestic animals as reservoirs of the human-infective Trypanosoma brucei gambiense.MethodBlood samples were collected from 155 donkeys and 131 horses in three human African trypanosomiasis (HAT) foci in Chad. Rapid diagnostic test (RDT) and capillary tube centrifugation (CTC) test were used to search for trypanosome infections. DNA was extracted from each blood sample and different trypanosome species and subspecies were identified with molecular tools.ResultsFrom 286 blood samples collected, 54 (18.9%) and 36 (12.6%) were positive for RDT and CTC, respectively. PCR revealed 101 (35.3%) animals with trypanosome infections. The Cohen’s kappa coefficient used to evaluate the concordance between the diagnostic methods were low; ranging from 0.09 ± 0.05 to 0.48 ± 0.07. Trypanosomes of the subgenus Trypanozoon were the most prevalent (29.4%), followed by T. congolense forest (11.5%), Trypanosoma congolense savannah (4.9%) and Trypanosoma vivax (4.5%). Two donkeys and one horse from the Maro HAT focus were found with T. b. gambiense infections. No significant differences were observed in the infection rates of different trypanosomes between animal species and HAT foci.ConclusionsThis study revealed several trypanosome species and subspecies in donkeys and horses, highlighting the existence of AAT in HAT foci in Chad. The identification of T. b. gambiense in donkeys and horses suggests considering these animals as potential reservoir for HAT in Chad. The presence of both human-infective and human non-infective trypanosomes species highlights the need for developing joint control strategies for HAT and AAT.

Ultrafast microliter centrifuge platform for size‐based separation using novel omnidirectional spiral surface acoustic wave for point‐of‐care diagnostics

Lab‐on‐a‐chip diagnostic devices have long promised the future ability to provide point‐of‐care information on a patient’s medical conditions from a small blood sample. Within the blood, there are numerous functional entities, including exosomes, microRNA, large functional proteins within the plasma and indeed platelets and white blood cells that altogether can provide a great deal of details regarding a patient’s burden of disease and disorders.Despite years of investment and research, none have found a method to perform rapid and effective centrifugation and separation of cells from whole blood. Still today a key reason that diagnostics remains a laboratory‐based activity is the need for centrifugation and fractionation of whole blood with sample sizes of at least 10 mL (about the volume of a large lipstick case). With humans, this requires drawing blood from the arm, uncomfortable and not possible to be repeated numerous times. With small animals used in research, such separations are tedious and certainly require less than 10 mL of blood to avoid killing the animal.Here, we have devised a solution to the true problem of lab‐on‐a‐chip, point‐of‐care diagnostic devices—fluid handling at chip (micro to nano) scales—by using a carefully designed, sterilizable ~40 MHz surface acoustic wave microfluidics handling device that can centrifuge and fractionate whole blood using samples as small as 2 μL. This is a mere pinprick of blood from a human or animal.Acoustic waves transmitted into fluids at frequencies above 5 MHz can generate rapid fluid flows via a combination of compressibility and local fluid motion from the passage of the acoustic wave, but avoid damaging the cells and molecules in the sample as determined via proliferation, QPCR, and mass spectroscopy techniques among other methods. Placing a droplet of whole blood on our device allows it to be spun at over 3000 RPM to cause separation of the particulate components of the blood due to centrifugation and direct interaction of the acoustic wave with those particulates (i.e., the cells). With our unique device, we can furthermore rapidly reverse the rotation of the blood in a few milliseconds, producing an additional means to induce mixing and more finely controlled separation of objects in the blood. The centrifugation completes in ~1 s, about two orders of magnitude quicker than in an ultracentrifuge. After centrifugation, we extract the various components of the blood through microchannels strategically placed to deliver red blood cells, platelets, and other cells separated from each other. These advancements potentially provide an effective platform for whole blood separation and point‐of‐care diagnostics without need for micro or nanoscale fluidic enclosure.Image of 40MHz Omnidirectional spiral surface acoustic wave device with microliter sessile drop on inner circular region. Scale bar: 0.5mm.Figure 1(a) 1.5 μl drop with 3.3μm (red), 5.73μm (green), 43.2μm (green) polystyrene (PS) particles randomly distributed inside. (b) After surface acoustic wave induced separation, showing 3.3μm, 5.73μm, 43.2μm PS particles separated from periphery to center of the droplet. (c) 1.5 μl diluted mouse blood before separation. (d) After surface acoustic wave induced separation, obtaining red blood cells at center and other components of blood at periphery of the drop. Scale bar: 0.5mm.Figure 2

Eicosapentaenoic Acid (EPA) has High Affinity for Cardiac Phospholipid Membranes Compared to other Triglyceride-Lowering Agents

Background: The benefits of EPA reported in REDUCE-IT appear to extend beyond TG-lowering. EPA is known to have membrane effects, and since it is unknown if other TG-agents exhibit like qualities we compared the binding of EPA to other TG-lowering agents in membranes derived from model and cardiac phospholipids. Distinct membrane interactions may contribute to pleiotropic benefits of EPA. Methods: The membrane binding or partition coefficient (Kp ) for EPA, fenofibric and nicotinic acid was measured by rapid centrifugation methods. Membrane vesicles were reconstituted from phosphatidylcholine (PC) extracted from bovine heart tissue or 1-palmitoyl-2-oleoyl-sn-glycero-3-PC (POPC) and cholesterol. Following centrifugation, samples were analyzed for drug concentrations using UV/Vis spectrophotometry. The Kp values are expressed as the mean ± SEM. Results: The measured Kp value for EPA was 6047 ± 1520 and approximately 8-fold higher than nicotinic acid and fenofibric acid which were 737 ± 30 and 763 ± 480, respectively. The Kp values in the cardiac membranes were consistent with those measured in model membranes composed of POPC and cholesterol: the Kp value for EPA was 7829 ± 1210 and again higher than nicotinic acid and fenofibric acid which were 468 ± 27 and 51 ± 20, respectively. Conclusion: As expected, these data suggest that EPA, unlike other TG-lowering agents, is able to partition efficiently into membranes with high affinity where it may exert pleiotropic effects beyond TG-lowering such as membrane stability and inhibition of lipid oxidation. These strong membrane interactions of EPA may contribute to atheroprotective benefits beyond TG-lowering.

Validation of a sensitive UHPLC-MS/MS method for cytochrome P450 probe substrates caffeine, tolbutamide, dextromethorphan, and alprazolam in human serum reveals drug contamination of serum used for research

To evaluate the potential for interactions between botanical dietary supplements and drug metabolism, Phase I clinical pharmacokinetics studies are conducted using an oral cocktail of probe substrates of cytochrome P450 (CYP) enzymes. A sensitive, specific, and fast ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for determination of caffeine (probe of CYP1A2), tolbutamide (probe of CYP2C9), dextromethorphan (probe of CYP2D6), and alprazolam (probe of CYP3A4/5) in human serum. Stable isotope-labelled analogs were used as internal standards, and sample preparation involved only rapid protein precipitation and centrifugation. The method of standard addition was used for the measurement of caffeine, because commercially available pooled human serum contains caffeine. Out of 18 lots of pooled human serum tested, caffeine was detection in all lots, alprazolam was detected in 13 lots, 8 lots contained dextromethorphan, and no tolbutamide was detected. Only serum prepared from the blood of select individuals was determined to be drug-free. The analytical method was validated with respect to linearity, accuracy and precision, recovery, stability, and matrix effects. The calibration curves were linear over the range of 25-12,000 ng/mL for caffeine, 75-36,000 ng/mL for tolbutamide, 0.05−30 ng/mL for dextromethorphan, and 0.1−60 ng/mL for alprazolam. The intra-assay and inter-assay coefficients of variation (%CV) and %Bias were <13 % (<17 % at the lower limit of quantitation). The recovery of each probe substrate ranged from 84.2%–98.5 %. All analytes were stable during sample storage and handling. Matrix effects were minimized by using stable isotope-labeled internal standards. The method was successfully applied to clinical studies investigating the pharmacokinetic alterations of probe substrates caused by chronic consumption of botanical dietary supplements.

The 3 R’s for Platelet-Rich Fibrin: A “Super” Tri-Dimensional Biomaterial for Contemporary Naturally-Guided Oro-Maxillo-Facial Soft and Hard Tissue Repair, Reconstruction and Regeneration

Platelet-Rich fibrin (PRF) is a three-dimensional (3-D) autogenous biomaterial obtained via simple and rapid centrifugation from the patient’s whole blood samples, without including anti-coagulants, bovine thrombin, additives, or any gelifying agents. At the moment, it is safe to say that in oral and maxillofacial surgery, PRFs (particularly, the pure platelet-rich fibrin or P-PRF and leukocyte and platelet-rich fibrin or L-PRF sub-families) are receiving the most attention, essentially because of their simplicity, cost-effectiveness, and user-friendliness/malleability; they are a fairly new “revolutionary” step in second-generation therapies based on platelet concentration, indeed. Yet, the clinical effectiveness of such surgical adjuvants or regenerative platelet concentrate-based preparations continues to be highly debatable, primarily as a result of preparation protocol variability, limited evidence-based clinical literature, and/or poor understanding of bio-components and clinico-mechanical properties. To provide a practical update on the application of PRFs during oral surgery procedures, this critical review focuses on evidence obtained from human randomized and controlled clinical trials only. The aim is to serve the reader with current information on the clinical potential, limitations, challenges, and prospects of PRFs. Accordingly, reports often associate autologous PRFs with early bone formation and maturation; accelerated soft-tissue healing; and reduced post-surgical edema, pain, and discomfort. An advanced and original tool in regenerative dentistry, PRFs present a strong alternative and presumably cost-effective biomaterial for oro-maxillo-facial tissue (soft and hard) repair and regeneration. Yet, preparation protocols continue to be a source of confusion, thereby requiring revision and standardization. Moreover, to increase the validity, comprehension, and therapeutic potential of the reported findings or observations, a decent analysis of the mechanico-rheological properties, bio-components, and their bioactive function is eagerly needed and awaited; afterwards, the field can progress toward a brand-new era of “super” oro-dental biomaterials and bioscaffolds for use in oral and maxillofacial tissue repair and regeneration, and beyond.

Comparison of Rapid Centrifugation Technique with Conventional Centrifugation for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) Testing.

Prothrombin Time (PT) and activated partial thromboplastin time (APTT) are frequently performed coagulation tests in patients with coagulation disorders especially in critical care areas and in monitoring patients on anticoagulation therapy. In coagulation testing, sample processing especially centrifugation is one of the most critical steps that affect turnaround time(TAT). This study was carried out over a period of 1year. Three hundred paired samples from patients sent for PT and APTT estimation were included. One sample was centrifuged in a regular bench top centrifuge at 1500g for 20min. The other sample was divided into two polypropylene aliquots and centrifuged in a microcentrifuge at 13000g for 3min. The plasma obtained from both methods was tested for PT and APTT using the automated method on STA Compact coagulometer (Stago) using commercial thromboplastin STAR-NeoplastineR C1 Plus and phospholipid (cephalin), STAR-C K PRESTR 5 respectively. Data were analyzed using descriptive statistics, Student t test, correlation coefficient and Bland-Altman plots. Mean PT, INR and APTT forboth centrifugation methods was comparable with no statistically significant difference (p > 0.05). PT, INR and APTT also showed good correlation (r > 0.98) when compared between the two methods of centrifugation. Bland-Altman comparison between rapid and conventional methods of centrifugation for PT, INR and APTT also showed acceptable agreement. Rapid centrifugation technique for routine coagulation testing can be used safely with a significant reduction in the TAT. This can benefit patients in critical care settings and those on outpatient oral anticoagulant therapy.

Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function.

Retroviral (RV) expression of genes of interest (GOIs) is an invaluable tool and has formed the foundation of cellular engineering for adoptive cell therapy in cancer and other diseases. However, monitoring of transduced T cells long term (weeks to months) in vivo remains challenging because of the low frequency and often poor durability of transduced T cells over time when transferred without enrichment. Traditional methods often require additional overnight in vitro culture after transduction. Moreover, in vitro-generated effector CD8+ T cells enriched by sorting often have reduced viability, making it difficult to monitor the fate of transferred cells in vivo. Here, we describe an optimized mouse CD8+ T-cell RV transduction protocol that uses simple and rapid Percoll density centrifugation to enrich RV-susceptible activated CD8+ T cells. Percoll density centrifugation is simple, can be done on the day of transduction, requires minimal time, has low reagent costs and improves cell recovery (up to 60%), as well as the frequency of RV-transduced cells (∼sixfold over several weeks in vivo as compared with traditional methods). We have used this protocol to assess the long-term stability of CD8+ T cells after RV transduction by comparing the durability of T cells transduced with retroviruses expressing each of six commonly used RV reporter genes. Thus, we provide an optimized enrichment and transduction approach that allows long-term in vivo assessment of RV-transduced T cells. The overall procedure from T-cell isolation to RV transduction takes 2 d, and enrichment of activated T cells can be done in 1 h.