Bystander apoptosis in human cells mediated by irradiated blood plasma

Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G(0)-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40Gy acute γ-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24h at 37°C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2±1.8% in plasma-free cultures, 21.6±1.1% in cultures treated with plasma from unirradiated blood, 20.2±1.4% in cultures with plasma from blood given 2-4Gy and 16.7±3.2% in cultures with plasma from blood given 6-10Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.     

Distribution of Abeta peptide in whole blood

The measurement of amyloid beta peptides (Abeta) in blood and plasma is expected to be a useful biomarker as potential therapeutics designed to lower Abeta peptide enter clinical trials. Many reports have suggested that Abeta could bind to substances in blood that may influence the recovery of Abeta peptide in plasma, its detection by conventional ELISAs or the actual turnover and half-life of the peptide in blood. In this study we describe a process for analyzing total Abeta in whole blood and plasma using denaturing solid-phase extraction followed by reverse-phase HPLC linked to ELISA. Comparison of total Abeta peptide levels in whole blood and plasma from the same bleed showed that most of the Abeta peptide is captured in the plasma if the samples are first denatured. In contrast, plasma that was assayed without denaturation could show greater than 70% reduction in apparent total Abeta peptide. This suggested that there was a pool of Abeta peptide in non-denatured plasma that is occluded from detection by ELISA, perhaps by binding to plasma proteins.     

Proteomic analysis of microvesicles from plasma of healthy donors reveals high individual variability

Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeutic outcomes it is critical to control the quality of blood plasma. Clearly initiatives to improve the safety of blood transfusions will have a high economical and social impact. A detailed knowledge of the composition of healthy blood plasma is essential to facilitate such improvements. Apart from free proteins, lipids and metabolites, blood plasma also contains cell-derived microvesicles, including exosomes and microparticles from several different cellular origins. In this study, we have purified microvesicles smaller than 220nm from plasma of healthy donors and performed proteomic, ultra-structural, biochemical and functional analyses. We have detected 161 microvesicle-associated proteins, including many associated with the complement and coagulation signal-transduction cascades. Several proteases and protease inhibitors associated with acute phase responses were present, indicating that these microvesicles may be involved in these processes. There was a remarkably high variability in the protein content of plasma from different donors. In addition, we report that this variability could be relevant for their interaction with cellular systems. This work provides valuable information on plasma microvesicles and a foundation to understand microvesicle biology and clinical implications.     

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes¹, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes^2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.     

Transfer of excess cholesterol from human red blood cells to plasma in vitro

This study examined the ability of plasma and plasma fractions from normolipidaemic subjects and plasma from a patient with homozygous familial high density lipoprotein deficiency (Tangier disease) to promote loss of excess cholesterol from red blood cells in vitro. Isolated high density lipoproteins were the most potent plasma fraction for removing excess cellular cholesterol. Lipoprotein-deficient plasma and human serum albumin, but not very low density lipoproteins and low density lipoproteins, also removed excess cholesterol from the red blood cells. The near absence of high density lipoproteins in plasma from the patient with Tangier disease did not result in an abnormally low rate of cholesterol loss from the enriched red blood cells. These results suggest that normal levels of high density lipoproteins are not vital for the removal of excess cholesterol from red blood cells by plasma.     

The rapid disappearance of muscle AMP aminohydrolase from blood plasma of normal and dystrophic chickens

The levels of creatine kinase and pyruvate kinase are increased 22 and 9.3 fold respectively in the blood plasma of dystrophic chickens as compared to normal controls. AMP aminohydrolase levels are not increased despite their abundance in muscle tissue. When AMP aminohydrolase was injected into a blood vessel, its rate of disappearance from the plasma was rapid with 97% of the enzyme disappearing with a half-life of 3.3 minutes. In contrast, the rate of disappearance of pyruvate kinase from the blood plasma is relatively slow, following a biphasic exponential decay with half-lives of 113 min and 710 min. These data suggest that the rates of disappearance of enzymes from the blood plasma is an important factor in determining whether increased plasma levels of these enzymes are observed in muscular dystrophy.     

In vitro metabolism of selenite in sheep blood: factors controlling the distribution of selenium and the labelling of plasma protein.

Some factors controlling the distribution of Na275SeO3 in sheep blood were studied in vitro. After centrifuging Na275SeO3-incubated blood most of the radioactivity was found in the plasma. The labelling of plasma protein by 75Se was dependent on the presence of erythrocytes. The degree of labelling of plasma protein increased with erythrocyte concentration. When phosphate-buffered saline-washed erythrocytes were suspended in phosphate-buffered saline and incubated with Na275SeO3 the majority of the 75Se was detected in the erythrocytes. On incubating these labelled erythrocytes with unlabelled plasma there was a transfer of radioactivity to the plasma. The calculated activation energy for the labelling of plasma was 107.52 kJ/mol. Albumin was shown not to be a principal acceptor of 75Se from the erythrocytes by ammonium sulphate precipitation of radioactive plasma. Addition of Na2SeO3 to the labelled blood resulted in the transfer of 75Se from plasma to the erythrocytes. Radioactive plasma incubated at 37 degrees C was thermolabile with respect to its 75Se content whereas in whole blood the degree of 75Se binding to plasma protein did not vary suggesting that a recycling of selenium was occurring in blood. From the results presented an in vitro model of selenium metabolism in blood is postulated.     

A refined blood collection method for quantifying corticosterone

In many rodent laboratories, blood samples are collected from rats using the tail vein nick procedure and analyzed to quantify blood corticosterone levels as an indicator of stress. The standard method of corticosterone quantification often requires the collection of a relatively large volume of blood, followed by the extraction of the blood plasma. An alternative blood sampling method requires the collection of only a drop of blood on paper (the 'drop' method), minimizing handling of the animals, and does not require plasma extraction. The authors aimed to validate the drop method of blood sampling for use in corticosterone quantification. They induced stress in rats by cerebral ischemia, collected blood samples at various intervals using both the drop method and the plasma extraction method and then quantified corticosterone by radioimmunoassay. Corticosterone levels of the ischemic rats were compared with those of sham-operated rats and those of ischemic rats that had been given metyrapone, a glucocorticoid synthesis inhibitor, prior to vessel occlusion. Blood corticosterone levels in the samples obtained from the same animal using the two different methods were highly correlated for all rats. The authors further provide a regression model that can be used to predict plasma corticosterone values from those obtained from the drop blood samples. Quantification of corticosterone from only a small drop of blood has many practical and ethical advantages and should be considered as an alternative to standard methods.     

Factors affecting sedimentational separation of bacteria from blood

Rapid diagnosis of blood infections requires fast and efficient separation of bacteria from blood. We have developed spinning hollow disks that separate bacteria from blood cells via the differences in sedimentation velocities of these particles. Factors affecting separation included the spinning speed and duration, and disk size. These factors were varied in dozens of experiments for which the volume of separated plasma, and the concentration of bacteria and red blood cells (RBCs) in separated plasma were measured. Data were correlated by a parameter of characteristic sedimentation length, which is the distance that an idealized RBC would travel during the entire spin. Results show that characteristic sedimentation length of 20 to 25 mm produces an optimal separation and collection of bacteria in plasma. This corresponds to spinning a 12-cm-diameter disk at 3,000 rpm for 13 s. Following the spin, a careful deceleration preserves the separation of cells from plasma and provides a bacterial recovery of about 61 ± 5%.     

Leukaemic peripheral blood plasma and bone marrow plasma: comparison of influence on lymphocyte proliferation

Abstract. Peripheral blood plasma from some children with untreated acute lymphoblastic leukaemia (ALL) exerted an inhibitory effect in vitro on phytohaemagglutinininduced lymphocyte transformation of normal peripheral blood lymphocytes. This occurred at concentrations beyond that required for optimal response as judged by reduction of blast cell formation and tritiated thymidine and tritiated uridine incorporation into DNA and RNA, respectively. In contrast, bone marrow plasma from these patients was non-inhibitory or contained significantly less inhibitory activity. Bone marrow plasma from the majority of healthy controls was superior to their peripheral blood plasma in enhancing phytohaemagglutinin-induced mitogenesis. The difference between an individual's bone marrow- and peripheral blood-derived plasma in enhancing proliferation of patient and healthy control cells was significantly greater amongst the patients than the healthy control group; this was attributed mainly to the increased inhibitory activity of ALL peripheral blood plasma compared with normal plasma. Medium conditioned by phytohaemagglutinin-stimulated normal peripheral blood lymphocytes was effective in neutralizing the inhibitory activity of ALL peripheral blood plasma. Taken together, these in vitro results are at least suggestive that in vivo , in healthy subjects, the rapidly proliferating cells in the bone marrow and the 'resting' blood cells in the circulation may be under the influence of a fine balance of different types and/or levels of humoral growth stimulatory and inhibitory factors and that in ALL an unstable balance of these factors exists. The decreased proliferation of circulating blast cells compared with bone marrow blasts in ALL may be attributed, at least in part, to exposure to the different levels of inhibitor(s) in the circulation and bone marrow as demonstrated in vitro by our results.     

Effect of blood withdrawal and angiotensin II on prolactin release in the tilapia,Oreochromis mossambicus

Repeated blood withdrawal (5% of estimated blood volume at 0, 1, 4, 8, 24, 48 and 76 h) from tilapia acclimated to fresh water (FW) resulted in a marked increase in plasma levels of prolactin (PRL) during the first 8 h, reaching a peak above 300 ng/ml after 4 h. The increase in plasma PRL levels was significant except for the level after 72 h. A slight but significant decrease in plasma osmolality was observed at all time points after the blood withdrawal. Repeated blood withdrawal from fish acclimated to seawater (SW) resulted in a marked increase in plasma osmolality after 4 and 8 h. A significant increase was observed in plasma growth hormone (GH) in the fish in SW until the end of the experiment, but there was no change in plasma PRL. Plasma levels of cortisol were significantly higher in the fish in SW than in those in FW during the first 24 h. Blood withdrawal resulted in a significant reduction in hematocrit values in both FW- and SW-adapted fish, suggesting hemodilution. In a separate experiment, a single blood withdrawal (20% of total blood) stimulated drinking after 5 h, regardless of whether the fish were held in FW or SW. Plasma PRL level was also elevated following a single blood withdrawal in the fish acclimated to FW, but not in the fish in SW. Intraperitoneal injection of ANG II (1.0 microg/g) into the fish in FW significantly increased plasma PRL levels after 1 h. Activation of the renin-angiotensin system after blood withdrawal and the dipsogenic action of angiotensin II (ANG II) are well established in fish. The reduction in plasma osmolality after repeated blood withdrawal in FW and the increased osmolality in SW suggest that blood volume is restored, at least in part, by drinking environmental water. These results suggest that the marked increase in PRL concentration after blood withdrawal from the fish in FW is due, at least in part, to a facilitative effect between ANG II and reduced plasma osmolality.     

The efficacy of field techniques for obtaining and storing blood samples from fishes

Prompted by the dramatic increase in the use of blood analyses in fisheries research and monitoring, this study investigated the efficacy of common field techniques for sampling and storing blood from fishes. Three questions were addressed: (1) Do blood samples taken via rapid caudal puncture (the ‘grab‐and‐stab’ technique) yield similar results for live v. sacrificed groups of fishes? (2) Do rapidly obtained caudal blood samples accurately represent blood properties of fishes prior to capture? (3) Does storage of whole blood in an ice slurry for a working day (8·5 h) modify the properties of the plasma? It was shown that haematocrit, plasma ions, metabolites, stress hormones and sex hormones of caudal blood samples were statistically similar when taken from live v. recently sacrificed groups of adult coho salmon Oncorhynchus kisutch. Moreover, this study confirmed by using paired blood samples from cannulated O. kisutch that blood acquired through the caudal puncture technique (mean ±s.e . 142 ± 26 s after capture) was representative of fish prior to capture. Long‐term (8·5 h) cold storage of sockeye salmon Oncorhynchus nerka whole blood caused significant decreases in plasma potassium and chloride, and a significant increase in plasma glucose. Previous research has suggested that these changes largely result from net movements of ions and molecules between the plasma and erythrocytes, movements that can occur within minutes of storage. Thus, blood samples from fishes should be centrifuged as quickly as practicable in the field for separation of plasma and erythrocytes to prevent potentially misleading data.     

The stimulation of sheep monocyte mitosis in vitro by autologous plasma taken after surgery and the implantation of foreign material.

When blood monocytes from sheep are grown upon glass in 100% autologous plasma they divide with low mitotic indices, but if the plasma is taken from the sheep three or four days after the implantation of a small Teflon or stainless steel cylinder, the maximum mitotic indices of the monocytes cultured in that plasma rise. Plasma collected from the sheep in the first two days after operation is often toxic to cultured autologous monocytes. An equivalent sham operation increases the mitogenicity of the plasma, but without a preceding toxic period. No consistent effects upon the numbers of circulating blood monocytes were noted following implantation operations. A hypothesis is presented which suggests that blood monocyte multiplication in vivo and in vitro is dependent upon (a) a lesion which causes cell attachment, and (b) a humoral component of the plasma which stimulates mitosis. Surgery, and the implantation of foreign materials will increase the concentration of such a substance in plasma.     

Direct determination of selenium in human blood plasma and seminal plasma by graphite furnace atomic absorption spectrophotometry and clinical application

Direct determination of selenium (Se) in body fluids by graphite furnace atomic absorption spectrophotometry (GFAAS) may suffer from problems like severe background, matrix effects, preatomization losses, and spectral interferences. In this study we evaluate critically the influence on the accuracy of the direct determination of Se in blood plasma and seminal plasma by GFAAS, and propose a simple, rapid, and accurate method, suitable for routine clinical analysis. The method for blood plasma is mainly based on studies by the use of matched matrix and a Pd-Ni modifier, but for seminal plasma only a Pd modifier is required. The method developed was also applied to study the Se distribution in plasma protein fractions of patients with hepatocellular carcinoma. The Se in plasma of patients was significantly lower than that of the controls. The distribution pattern of Se in blood plasma fractions of patients was also different from that of the controls.     

Substance P in the blood plasma in hypophysial portal vessels

The aim of the present study was to check if Substance P (SP) is released from the hypothalamus into the hypophysial portal vessels and by this route exerts its direct influence on the adenohypophysis. For this purpose SP radioimmunoactivity was assayed in the blood plasma collected from hypophysial portal vessels and from the cephalic end of the external jugular vein. The SP levels in blood plasma collected from hypophysial portal vessels and from the jugular vein do not differ significantly. Neither does application of a noxious factor, such as bilateral femoral bone fracture, change significantly the SP level in the blood plasma from portal vessels and from the jugular vein. Hypoxia seems to increase the SP level in portal blood plasma and may be followed by its decrease. It is concluded that hypothalamic SP is not released into the hypophysial portal vessels under normal conditions and its direct influence on the adenohypophysis is not mediated this way.     

TSE clearance during plasma products separation process by Gradiflow(TM).

BACKGROUND AND OBJECTIVES: Recent experimental evidence from rodent models suggests a potential risk for transmissible spongiform encephalopathy (TSE) transmission by blood. The emergence of a new variant Creutzfeldt-Jakob disease (vCJD) has raised increased concerns about the safety of blood components and plasma products derived from vCJD-infected donors. Recent risk-minimisation strategies have included a ban on the use of UK-sourced plasma for the preparation of licensed blood products and leukodepletion of blood donations for fear of possible transmission of the human TSE via blood or blood components. The aim of this study was to investigate the capability and efficacy of a preparative electrophoresis system (Gradiflow) in the removal of TSE contaminants during the separation of plasma products. MATERIALS AND METHODS: Using hamster adapted scrapie 263 K as a model for TSE agent, albumin and IgG separation from human plasma by Gradiflow were performed separately by spiking a 263 K scrapie microsomal fraction to the feed material at each process step. Samples from pre- and post-Gradiflow separation process were titrated to the end-point for the detection of the disease-associated, proteinase K resistant form of the pathogenic prion protein (PrP(Sc)) by Western blot. RESULTS: Under all conditions tested, a greater than 3 log(10) reduction was achieved with no PrP(Sc) detected in any of the pooled products for either of the IgG or albumin separations. These data show that Gradiflow processing has clear advantages for concurrent purification of plasma products and in-process TSE removal. CONCLUSIONS: Our findings suggest that Gradiflow process is a viable alternative to remove causative TSE agents during plasma products separation, potentially eliminating the risk of TSE agents transmission.     

Effect of dilution on sedimentational separation of bacteria from blood

Bacteria must be separated from septic whole blood in preparation for rapid antibiotic susceptibility tests. This work improves upon past work isolating bacteria from whole blood by exploring an important experimental factor: Whole blood dilution. Herein, we use the continuity equation to model red blood cell sedimentation and show that overall spinning time decreases as the blood is diluted. We found that the bacteria can also be captured more efficiently from diluted blood, up to approximately 68 ± 8% recovery (95% confidence interval). However, diluting blood both requires and creates extra fluid that end users must handle; an optimal dilution, which maximizes bacteria recovery and minimizes waste, was found to scale with the square root of the whole blood hematocrit. This work also explores a hypothesis that plasma backflow, which occurs as red cells move radially outward, causes bacterial enrichment in the supernatant plasma with an impact proportional to the plasma backflow velocity. Bacteria experiments carried out with diluted blood demonstrate such bacterial enrichment, but not in the hypothesized manner as enrichment occurred only in undiluted blood samples at physiological hematocrit.     

Haemolysis during sample preparation alters microRNA content of plasma

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.     

Fatty acid composition of plasma, blood cells and whole blood: relevance for the assessment of the fatty acid status in humans

The composition and incorporation of fatty acids (FA) in plasma and blood cells is the result of distinct processes: intake, metabolism and peripheral utilization. Aim of the study: was to compare the FA profile in plasma, lipoproteins and blood cells with that in whole blood (WB) from healthy volunteers; to assess the quantitative distribution of selected FA in triacylglycerols, cholesteryl esters and phospholipids. Lipid FA profiles are comparable in plasma and lipoproteins but differ from those in blood cells. In WB, the FA profile results from the balanced proportion of FA pools in plasma and cells. The contribution of each lipid class to the total amount of FA differs among blood specimens. Phospholipids of plasma and red blood cell are the major contributors to the FA amount and profile in WB. In conclusion, the FA profile of WB reflects the FA status and WB could be an adequate specimen for the assessment of FA intakes.     

Optimization of Conditions for Blood Plasma Peptidome Analysis

The conditions for peptidome analysis of blood plasma were optimized and the efficacy of the proposed approach was compared with the methods described in the literature. The method implies solution of two main problems: inactivation of blood plasma proteases and dissociation of peptides from major blood plasma proteins, which they are quantitatively associated with. To solve these problems, we proposed a new method of sample preparation. The essence of the method is simultaneous denaturation of plasma proteins plus reduction and alkylation of thiol groups of Cys, which is achieved by heating a blood plasma sample (95°C) in the presence of sodium deoxycholate, tris(2-carboxyethyl)phosphine, and 2-chloroacetamide. After separation of peptides from proteins by ultrafiltration on microcentrifuge filters and removal of sodium deoxycholate, the peptides are identified by LC-MS/MS using a Q Exactive HF (Thermo Scientific) mass spectrometer. As a result of one LC-MS/MS run of the peptide mixture obtained from ~15 μL of blood plasma, 2257 peptide fragments of 867 proteins were identified, which is 1.5 times higher than the values achieved by using the generally accepted method of differential solubilization. Our immediate plans include the use of our approach for cataloguing human blood plasma peptides, as well as establishing the magnitude of individual variability and the features of the peptidome that are related to gender and age.