Consecutive Aliquots Research Articles

Stabilization of Human Whole Blood Samples for Multicenter and Retrospective Immunophenotyping Studies.

Whole blood is often collected for large-scale immune monitoring studies to track changes in cell frequencies and responses using flow (FC) or mass cytometry (MC). In order to preserve sample composition and phenotype, blood samples should be analyzed within 24 h after bleeding, restricting the recruitment, analysis protocols, as well as biobanking. Herein, we have evaluated two whole blood preservation protocols that allow rapid sample processing and long-term stability. Two fixation buffers were used, Phosphoflow Fix and Lyse (BD) and Proteomic Stabilizer (PROT) to fix and freeze whole blood samples for up to 6months. After analysis by an 8-plex panel by FC and a 26-plex panel by MC, manual gating of circulating leukocyte populations and cytokines was performed. Additionally, we tested the stability of a single sample over a 13-months period using 45 consecutive aliquots and a 34-plex panel by MC. We observed high correlation and low bias toward any cell population when comparing fresh and 6months frozen blood with FC and MC. This correlation was confirmed by hierarchical clustering. Low coefficients of variation (CV) across studied time points indicate good sample preservation for up to 6months. Cytokine detection stability was confirmed by low CVs, with some differences between fresh and fixed conditions. Thirteen months regular follow-up of PROT samples showed remarkable sample stability. Whole blood can be preserved for phenotyping and cytokine-response studies provided the careful selection of a compatible antibody panel. However, possible changes in cell morphology, differences in antibody affinity, and changes in cytokine-positive cell frequencies when compared to fresh blood should be considered. Our setting constitutes a valuable tool for multicentric and retrospective studies. © 2020 International Society for Advancement of Cytometry.

The effect of a single consecutive volume aspiration on concentrated bone marrow from the proximal humerus for clinical application

BackgroundLow aspiration volumes have been recommended to allow for higher concentrations of progenitor cells during bone marrow harvesting. However, these guidelines then require multiple aspiration attempts in order to maximize cellular yield. The purpose of this study was to investigate the effect of a single, high-volume aspiration with four consecutive aliquots on the number of nucleated cells (NCs) and colony-forming units (CFUs) in concentrated bone marrow aspirate (cBMA) taken from the proximal humerus.MethodscBMA was taken from the proximal humerus of patients undergoing arthroscopic rotator cuff surgery. Four 12-mL double syringes were used consecutively from a single trocar to obtain four 10 cc aliquots. Each then underwent centrifugation to create a fractionated layer rich in nucleated cells. Following cellular separation, NCs were counted and CFUs were evaluated after incubation of 7–10 days. Cellular comparisons between each aliquot were performed along with their interaction with patient age and sex.ResultsTwenty-nine patients (55.9 ± 4.6 years) were included in this study. The number of NCs and CFUs showed significant differences between the four aliquots of aspirate, with the first 10 cc aliquot providing the highest amount (p < 0.001, respectively). No significant differences were found between the sum of the three sequential aliquots compared to the initial 10 cc sample. There were no significant differences between male and female patients (p > 0.05). Increasing age resulted in no significant decrease in the number of NCs and CFUs across the four consecutive aliquots (p > 0.05).ConclusionIn conclusion, while the initial aliquot provided the greatest number of nucleated cells and cultured CFUs, the addition of each sequential volume aspirate yielded similar amounts in total. This demonstrates the potential effectiveness of obtaining of higher volume aspirates from the proximal humerus during rotator cuff repair.

Olfactory Discrimination in Adult Male Rats Undernourished during the Pre- and Neonatal Period

In the neonatal rat, the olfactory system is a fundamental channel to locate and recognize the mother, to survive and develop early social behavior, and to distinguish related or unrelated members of a litter. Perinatal undernutrition in rats alters the neuronal organization and functioning of the olfactory system and its ascending relays, possibly affecting odor discrimination. In this study we compared the frequency and time spent visiting the source of urine obtained from unrelated female subjects and amyl acetate odors vs water in adult perinatally underfed rats. Each test initiated with one daily presentation during three consecutive aliquots of deionized water (10 μl) placed on a filter paper for 2 min, separated by 1-min intervals for habituation. The water presentations were followed by three exposures to an odor, all at the same dilution (either 1:1 or 1:80) for dishabituation. Control (C) and undernourished (U) subjects display similar, significant habituation/dishabituation responses, except that the U subjects showed greater increases in the frequency and duration of visits to undiluted and diluted urine and amyl acetate cues compared to the controls. These findings are similar to previous studies showing that adult rats investigate novel odors longer than the familiar ones. Thus, perinatal undernutrition appears to interfere with the foundation of the olfactory development causing long-term olfactory discrimination deficits as revealed by the increased frequency and duration of visits to the source of odors that may be relevant for social behavior.

In vitro bactericidal efficacy of a new sun- and heat burn gel

We assessed the in vitro bactericidal efficacy of a new sunburn gel (Rescudermtrade mark; RESC) against planktonic and sessile Pseudomonas aeruginosa (PSEUD) and Staphylococcus epidermidis (STAPH). While PSEUD levels were 4log(10) lower than those of STAPH within 24h of adding RESC to contaminated nutrient broths, all bacterial counts were comparable by 48h. PSEUD and STAPH levels were then measured after applying either a single or three consecutive aliquots of RESC to polyurethane sponges. Gel was removed after 5 or 20min, or left on for 72h. Bacterial counts in placebo-treated sponges had plateaued by 24h to values above 9log(10)CFU/mL. In contrast, six out of seven of the RESC application modalities reduced bacterial levels below 4log(10)CFU/mL for 72h. RESC remained effective against STAPH despite up to a 24h treatment delay, irrespective of the number of applications. Repeated RESC applications were required to maintain PSEUD below 4log(10)CFU/mL when the delay exceeded 7h. These data demonstrate the differential susceptibility of planktonic and sessile bacteria to RescuDermtrade mark. This product might be a good candidate for reducing the opportunity for wound infection, especially in burns.

Antithrombin III Binding to Surface Immobilized Heparin and Its Relation to F Xa Inhibition

The mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depolymerized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 and 11 picomoles/cm2, respectively. Thus the estimated density of biologically active high and low affinity heparin respectively would be 40 and 90 ng/cm2. The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. It was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromised AT binding sequences.

A sequential broncho-alveolar washing in non-anaesthetized normal bovines: Method and preliminary results

The method of lung lavage under fiberoptic control allowed collection of alveolar cells in non-anaesthetized adult cows. The median section of the diaphragmatic lobe was lavaged with five consecutive aliquots of 30 ml each. Every one was analysed separately. A mean of 25.6% of instilled fluid was recovered and this is lower than amounts obtained on isolated lungs or in anaesthetized calves (about 50%). The cellular formula of 30 samples showed 83.5% of macrophages, 6.0% of lymphocytes, 9.4% of polymorphonuclear cells, 0.5% of monocytes. Cellular viability and total cell count were similar to previously published data. All results were found to be independent of the washing sequence. This simple and well tolerated technique appeared to be a useful tool for the study of defence mechanisms of deep lung.

A high-precision mass spectrometer for stable carbon isotope analysis at the nanogram level

Details are presented of a double-collector static mass spectrometer that can determine precise stable carbon isotope ratios for samples of the order of 10-8 g carbon as CD4. Measurements are performed by analysis of the ion-beam intensities at m/z=20 (12CD4+)and 21 (13CD4+). The instrument is capable of long-term reproducibility (the 20/21 ratio of 23 consecutive aliquots of standard gas analysed over the course of a day varies by only +or-0.16 per mille); its ability to provide comparative isotopic measurements ( delta values) has been assessed by a zero-enrichment check which affords a value of -0.012+or-0.25 per mille.

Stimulated secretion of esterified lipids and VLDL by perifused hepatocytes.

This report describes a new method for investigation of hepatic metabolism by perifusion of medium through a batch of hepatocytes isolated from fed rats. Oxygenated medium flows by gravity through a hepatocyte-containing glass column that is immersed in 37 degrees C water bath. The effluent medium is then collected in consecutive aliquots in test tubes on ice. The pattern of export of esterified lipids, glucose, and VLDL by isolated liver cells into the perifusate was examined under both basal conditions and response to the infusion of certain metabolic stimulants. Perifusion of medium containing sodium clofibrate (1 mM and 10 mM levels) through lipid-prelabeled cells augmented the secretion of radioactive triacylglycerols that reached a maximal rate by about 30 min after exposure to this agent. Measurement of effluent glucose levels after perifusion of hepatocytes with media lacking glucose but containing a gluconeogenic precursor revealed steadily declining concentrations despite the addition of glucagon or epinephrine. Concomitantly, glycogen granules disappeared from the cytoplasm, but the cells retained intact ultrastructure after the course of perifusion. Protein-prelabeled hepatocytes released labeled VLDL into the perifusate, and this release was enhanced by prolonged exposure of the cells to medium containing palmitate (0.80 mM). The hepatocyte perifusion system thus offers a simple, reproducible method whereby hepatocellular secretory processes can be sequentially examined under carefully controlled basal and stimulated conditions.

Glutamine Phosphoribosylpyrophosphate Amidotransferase

Publisher Summary Glutamate assays are compromised by the presence of glutaminase activity in crude preparations, but this is not a problem with purified preparations of amidotransferase. Assay of pyrophosphate is insensitive, but this assay has been used with addition of a basal level of inorganic pyrophosphate. Assay of phosphoribosylamine is complex and involves preparation of other enzymes. Only the spectrophotometric assay of glutamate with glutamate dehydrogenase and 3-acetylpyridine nicotinamide adenine dinucleotide (3-AcPyNAD) lends itself to continuous kinetic measurement of activity. It has been found that the initial phases of the PP-ribose-P amidotransferase reaction are non-linear when determined with enzymatic or radiochemical assays of glutamate in consecutive aliquots of the reaction mixture. The lag of the reaction reflects the conversion of inactive to active enzyme by PP-ribose-P and Mg2+.