Effect of pre-analytical handling on haematological variables in minipigs

Pre-analytical handling may be an important determinant of haematological variables, if analysis is delayed. We investigated the effect of anticoagulants, i.e. tripotassium ethylenediamine-tetraacetic acid (EDTA) and citric acid, theophylline, adenosine, dipyridamole (CTAD), storage time (0.5, 1.5, 3.5, 5.5, 7.5, 25.5 and 27.5 h after blood sampling), and storage temperature (5 degrees C and 20 degrees C) on the variation in haemoglobin (HGB), red blood cell count (RBC), haematocrit (HCT), white blood cell count (WBC), and platelet count (PLT) in minipigs. Medians of HGB, RBC, HCT, WBC and PLT were significantly higher in EDTA tubes than in CTAD tubes due to the dilution effect of the anticoagulant. We found a minor significant increase in HCT after 25.5 h in blood stored at 20 degrees C, and at the same time a minor significant increase in WBC in EDTA tubes stored at 20 degrees C. We found a significant decrease in PLT in blood stored at 5 degrees C, especially in EDTA tubes. Minor variations were also observed in HGB and RBC. Our results indicate that PLT should only be measured in tubes placed at room temperature. If HCT or WBC analyses are to be performed on the day after blood sampling, the samples must be stored in a refrigerator until analysis. Our studies underline that time delay before analysis of haematological variables can cause increased variation, and should therefore be limited as far as possible in order to reduce the number of animals needed to make reliable conclusions.     

Immobilization of bacteria in silica matrices using citric acid in the sol–gel process

The aim of this work was to use citric acid in the sol–gel process to generate an inorganic polymer that allows bacterial survival for long periods of time and to study the influence of different storage temperatures. We compared gram-negative Escherichia coli and gram-positive Staphylococcus aureus, immobilized and preserved at different storage temperatures in silica matrices prepared by the method proposed. Immobilized E. coli and S. aureus in silica matrices were stored in sealed tubes at 20, 4, −20, and −70°C for 4 months during which the number of viable cells was analyzed. Results show that the immobilization in silica matrices using citric acid, to neutralize the alkalinity of the silica precursors, makes the technique not only biocompatible but also easier to perform since polymerization does not occur immediately as it does when hydrochloric acid is utilized.     

The effect of anticoagulants on Blennius pholis L. Leucocytes

The effects of ethylenediamine-tetraacetic acid (EDTA), heparin and tri-sodium citrate (TSC) on various haematological parameters in the blenny, Blennius pholis, were investigated. EDTA and heparin, at the concentrations tested, proved adequate in preventing coagulation although leucocyte viability was reduced when the higher levels were used. TSC failed to prevent coagulation at all concentrations tested. Short (5 min) and long (20 min) term contact of these anticoagulants with blood did not produce any significant changes in leucocyte proportions. Heparin is regarded as the most suitable anticoagulant for use with B. pholis blood.     

The influence of some sample handling factors on progesterone and testosterone analysis in goats

This study validated the use of commercially available radioimmunoassay kits for measuring the circulating progesterone and testosterone levels of goats. Progesterone and testosterone levels were then assayed in plasma which was collected from 23 does and 8 bucks. Collections from each animal were divided into three sodium fluoride-potassium oxalate (F/OX), one heparin, and one EDTA tubes and also into a tube without anticoagulant. Plasma from an F/OX tube was separated immediately from the blood cells by centrifugation. Serum or plasma was also separated after storage for 24 hours with F/OX, heparin or EDTA anticoagulant at 22 degrees C or with F/OX at 5 degrees C. A significant decline in assayable progesterone occurred in samples stored at 22 degrees C with each anticoagulant used and in the serum sample. Samples stored at 5 degrees C for 24 hours with F/OX anticoagulant contained concentrations of progesterone which did not differ significantly from those in samples where plasma was removed immediately. Assayable testosterone did not change with the anticoagulant used or vary with the storage temperature when F/OX tubes were stored at 5 degrees C and 22 degrees C for 24 hours. Results indicate that sample storage does influence levels of measured progesterone but not testosterone in goats. Progesterone assay is best done on plasma which is immediately separated from blood cells or on samples which are stored at 5 degrees C.     

Effects of sample handling on the stability of interleukin 6, tumour necrosis factor-alpha and leptin

Detected levels of IL-6, TNF-alpha and leptin may be affected by methods of storage, anticoagulant or repeated freezing-thawing. Blood samples from 22 healthy subjects were: (i) allowed to stand for 1, 2, 4 or 6 h prior to, or after separation, before freezing at -70 degrees C; (ii) taken into tubes with lithium heparin, sodium citrate, EDTA or no anticoagulant, separated and frozen; and (iii) separated, and plasma repeatedly freeze-thawed for up to six cycles prior to assay. Leptin was assayed by RIA, and IL-6 and TNF-alpha by high-sensitivity ELISA. (i) IL-6 and TNF-alpha levels were not altered significantly in separated samples but IL-6 declined by mean (SEM) 14.3% (3.7%) and TNF-alphaincreased by 9.6% (2.3%) in samples left unseparated for 4 h (P=0.003 and 0.002, respectively). Leptin remained unchanged. (ii) Serum and EDTA-plasma samples gave comparable results for all three cytokines, but levels in the other anticoagulant samples were highly variable. (iii) IL-6 and leptin levels were not altered by up to 6 cycles of freeze-thawing, but TNF-alpha increased by 17.0% (3.7%) after 3 cycles. Concentrations of these molecules are significantly altered by storage conditions, therefore they need to be standardized for epidemiological and clinical studies, and between-study comparisons of levels may not be reliable.     

Effects of anticoagulants on stable-isotope values (δ13C and δ15N) of shark blood components

The effects of anticoagulant EDTA and sodium heparin (SH) on stable carbon δ¹³C and nitrogen δ¹⁵N isotopic values of red blood cells (RBC) and blood plasma in juvenile blacktip reef sharks Carcharhinus melanopterus were analysed. Plasma preserved with anticoagulants was not isotopically distinct from plasma stored in no-additive control tubes but RBC δ¹⁵N values exhibited small enrichments when preserved with EDTA and SH. Results suggest EDTA and SH are viable anticoagulants for stable isotopic analyses of blood fractions but further studies are advised to validate results.     

The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

The effects of different decalcification protocols on TUNEL and general cartilage staining.

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

Effect of blood handling on extracellular Hsp72 concentration after high-intensity exercise in humans

Heat shock protein 72 (Hsp72) has been detected in the peripheral circulation of humans. Because intracellular Hsp72 binds to aggregated proteins, we hypothesized that postexercise plasma-derived Hsp72 concentrations would be greater than serum-derived Hsp72 because of binding of Hsp72 to aggregated clotting proteins in serum. Postexercise serum, heparin, and ethylenediaminetetraacetic acid (EDTA) samples were collected from 9 recreationally active males and were analyzed for Hsp72 by enzyme-linked immunosorbent assay. In line with our hypothesis, EDTA-treated blood was significantly higher in Hsp72 concentration than all other treatments (P < or = 0.001), whilst heparin plasma (LH) was significantly higher than serum derived on ice (SI) and at room temperature (SR) (P < 0.05; EDTA: 6.46 +/- 0.76, LH: 2.73 +/- 2.26, SI: 0.13 +/- 0.24, SR: 0.20 +/- 0.32 ng/mL). Because previous research has tended to report serum data at the lowest point of the detectable range of the assay, it is recommended that EDTA specimen tubes be used in future investigations.     

Enhanced phytoextraction: I. Effect of EDTA and citric acid on heavy metal mobility in a calcareous soil

Phytoextraction, the use of plants to extract heavy metals from contaminated soils, could be an interesting alternative to conventional remediation technologies. However, calcareous soils with relatively high total metal contents are difficult to phytoremediate due to low soluble metal concentrations. Soil amendments such as ethylene diaminetetraacetate (EDTA) have been suggested to increase heavy metal bioavailability and uptake in aboveground plant parts. Strong persistence of EDTA and risks of leaching of potentially toxic metals and essential nutrients have led to research on easily biodegradable soil amendments such as citric acid. In our research, EDTA is regarded as a scientific benchmark with which degradable alternatives are compared for enhanced phytoextraction purposes. The effects of increasing doses of EDTA (0.1,1,10 mmol kg(-1) dry soil) and citric acid (0.01, 0.05, 0.25, 0.442, 0.5 mol kg(-1) dry soil) on bioavailable fractions of Cu, Zn, Cd, and Pb were assessed in one part of our study and results are presented in this article. The evolution of labile soil fractions of heavy metals over time was evaluated using water paste saturation extraction (approximately soluble fraction), extraction with 1 M NH4OAc at pH 7 (approximately exchangeable fraction), and extraction with 0.5 M NH4OAc + 05 M HOAc + 0.02 M EDTA at pH 4.65 (approximately potentially bioavailable fraction). Both citric acid and EDTA produced a rapid initial increase in labile heavy metal fractions. Metal mobilization remained constant in time for soils treated with EDTA, but a strong exponential decrease of labile metal fractions was noted for soils treated with citric acid. The half life of heavy metal mobilization by citric acid varied between 1.5 and 5.7 d. In the following article, the effect of heavy metal mobilization on uptake by Helianthus annuus will be presented.     

Enhanced phytoextraction: II. Effect of EDTA and citric acid on heavy metal uptake by Helianthus annuus from a calcareous soil

High biomass producing plant species, such as Helianthus annuus, have potential for removing large amounts of trace metals by harvesting the aboveground biomass if sufficient metal concentrations in their biomass can be achieved However, the low bioavailability of heavy metals in soils and the limited translocation of heavy metals to the shoots by most high biomass producing plant species limit the efficiency of the phytoextraction process. Amendment of a contaminated soil with ethylene diamine tetraacetic acid (EDTA) or citric acid increases soluble heavy metal concentrations, potentially rendering them more available for plant uptake. This article discusses the effects of EDTA and citric acid on the uptake of heavy metals and translocation to aboveground harvestable plant parts in Helianthus annuus. EDTA was included in the research for comparison purposes in our quest for less persistent alternatives, suitable for enhanced phytoextraction. Plants were grown in a calcareous soil moderately contaminated with Cu, Pb, Zn, and Cd and treated with increasing concentrations of EDTA (0.1, 1, 3, 5, 7, and 10 mmol kg(-1) soil) or citric acid (0.01, 0.05, 0.25, 0.442, and 0.5 mol kg(-1) soil). Heavy metal concentrations in harvested shoots increased with EDTA concentration but the actual amount of phytoextracted heavy metals decreased at high EDTA concentrations, due to severe growth depression. Helianthus annuus suffered heavy metal stress due to the significantly increased bioavailable metal fraction in the soil. The rapid mineralization of citric acid and the high buffering capacity of the soil made citric acid inefficient in increasing the phytoextracted amounts of heavy metals. Treatments that did not exceed the buffering capacity of the soil (< 0.442 mol kg(-1) soil) did not result in any significant increase in shoot heavy metal concentrations. Treatments with high concentrations resulted in a dissolution of the carbonates and compaction of the soil. These physicochemical changes caused growth depression of Helianthus annuus. EDTA and citric acid added before sowing of Helianthus annuus did not appear to be efficient amendments when phytoextraction of heavy metals from calcareous soils is considered.     

Importance of calcium in citric acid-induced airway constriction

In previous studies, a 5-min inhalational challenge with 10% citric acid aerosol (0.52 M) elicited bronchoconstriction in Basenji-Greyhound (BG) dogs with hyperreactive airways but not in mongrel dogs. This response was independent of vagal reflexes because it was not attenuated by atropine. Citric acid might elicit bronchoconstriction because of acidity, calcium chelation, or some other effect of the citrate molecule. To assess these factors, barbiturate-anesthetized BG dogs were challenged (5 min) with aerosols of 10% acetic acid or a citric acid (0.48 M)/Na3citrate (0.04 M) mixture of equivalent pH, 6% Na2-ethylenediaminetetraacetic acid (EDTA), or 6% CaNa2EDTA. Each challenge was delivered in a separate week. The acidity alone was not an adequate stimulus, since pulmonary resistance (RL) was unaltered by 10% acetic acid, although markedly increased by the citric acid-Na3citrate mixture [2.2 +/- 0.4 (SE) cmH2O X l-1 X s prechallenge, 10.0 +/- 2.2 postchallenge]. Aerosols of Na2EDTA provoked a similar increase in RL (2.1 +/- 0.4 cmH2O X l-1 X s prechallenge, 9.0 +/- 1.8 postchallenge). Neither effect was attenuated by intravenous atropine (0.2 mg/kg). CaNa2EDTA caused no changes in RL. We conclude that it is the calcium chelating action of citric acid rather than its acidity that is responsible for bronchoconstriction in BG dogs with hyperreactive airways.     

Two methods for the prediction of Mycocentrospora acerina infection on stored carrots

Carrot plants were collected from 26 fields located in different districts in Norway during 1985, 1986 and 1987. Leaves were sampled in the first two years, and roots were sampled in all years. Incidence of Mycocentrospora acerina on the leaves at harvest was correlated (r = 0.82) to the incidence of M. acerina on the roots after storage at 0–1°C for six months (long term storage). Incidence of M. acerina on roots sampled about six weeks before harvest (date c) and placed at 10°C for six weeks (test storage) was correlated (r = 0.84) to incidence of the pathogen after long term storage on roots sampled at harvest (date d). Test storage and long term storage data from parallel samples taken at date d was more strongly correlated (r = 0.90).     

Decalcification for histochemical demonstration of hydrolytic and oxidative enzymes

Summary A method for histochemical demonstration of hydrolytic and oxidative enzymes following decalcification was described in mature bone and tooth by neutral EDTA decalcifying solution.The decalcifying solution, 0.5 M EDTA tetrasodium salt was adjusted to neutral pH with 5 M citric acid, obtained the most sufficient results for demonstration of enzymes in decalcified tissue. The hard tissue decalcified in 30 to 40 days at 4° C exhibited a good preservation of hydrolytic and oxidative enzymes histochemically, but a few structural destruction in a long term decalcification was found in soft tissues and certain organs.     

Impact of chelator-induced phytoextraction of cadmium on yield and ionic uptake of maize

Enhanced phytoextraction uses soil chelators to increase the bioavailability of heavy metals. This study tested the effectiveness of ethylenediaminetetraacetic acid (EDTA) and citric acid in enhancing cadmium (Cd) phytoextraction and their effects on the growth, yield, and ionic uptake of maize (Zea mays). Maize seeds of two cultivars were sown in pots treated with 15 (Cd₁₅) or 30 mg Cd kg^?1 soil (Cd₃₀). EDTA and citric acid at 0.5 g kg^?1 each were applied 2 weeks after germination. Results demonstrated that the growth, yield per plant, and total grain weight were reduced by exposure to Cd. EDTA increased the uptake of Cd in shoots, roots, and grains of both maize varieties. Citric acid did not enhance the uptake of Cd, rather it ameliorated the toxicity of Cd, as shown by increased shoot and root length and biomass. Cadmium toxicity reduced the number of grains, rather than the grain size. The maize cultivar Sahiwal-2002 extracted 1.6% and 3.6% of Cd from soil in both Cd+ EDTA treatments. Hence, our study implies that maize can be used to successfully phytoremediate Cd from soil using EDTA, without reducing plant biomass or yield.     

Effects of sampling conditions on selected blood variables of rainbow trout, Salmo gairdneri Richardson

The effects of two methods of specimen immobilization (MS 222 anaesthesia and stunning), two types of anticoagulant (EDTA and heparin), two storage temperature ranges (0–2°C and 22–25°C) and four sample storage periods (0, 1, 3, and 24 h) on the haemoglobin, haematocrit, plasma and packed cell sodium, potassium and chloride ion concentrations and packed cell ATP levels of rainbow trout were examined. Stored samples exhibited increases in cell volume, net transfer of sodium and chloride from plasma into cells, net loss of potassium to plasma and rapid depletion of ATP. Room temperature conditions and prolonged storage exacerbated these changes. Use of EDTA, particularly in combination with MS 222, frequently led to haemolysis. Least change in most variables was observed in samples drawn from stunned specimens, treated with heparin and refrigerated before use or preparation for deep cold storage.     

Effects of chronic hydrochloric and lactic acid administrations on food intake, blood acid-base balance and bone composition of the rat.

In experiment 1, weanling rats were given, for 7 weeks, a commercial rat diet supplemented with hydrochloric acid at levels up to 560 mmol.kg-1 dry matter. The supplement increased water intake but did not significantly affect food intake, live-weight gain, blood haemoglobin and haematocrit values or acid-base balance. In experiment 2, adult rats were given, for 9 weeks, a commercial rat diet supplemented with hydrochloric acid at levels up to 1250 mmol.kg-1 dry matter. Food intake and liveweight gain were not affected by hydrochloric acid concentration up to 625 mmole but at 938 mmol.kg-1 they were considerably reduced and there was 100% mortality of the rats. In experiment 3, weanling rats were given, for 12 weeks, a commercial rat diet supplemented with hydrochloric or lactic acid each at 300, 600 and 900 mmol.kg-1 dry matter. Lactic acid at the three levels and hydrochloric acid at the two lower levels did not affect food intake or live weight gain and had only a slight effect on blood acid-base balance. At a dietary concentration of 900 mmol.kg-1 dry matter, hydrochloric acid decreased food intake, induced a mild degree of metabolic acidosis and resulted in 30% mortality of the rats. In the three experiments, the acid treatments dnot directly affect the length or composition of the femur of the rats.     

Differential modulation of root formation in wheat embryogenic callus culture by indoleacetic acid-degrading compounds

Root overproduction in embryogenic calli of wheat cv. ‘ChineseSpring’ was controlled by the addition of IAA-degradingcompounds (citric acid, MnSO4 or EDTA) to the culture media.In the presence of 20 gl^–1 sucrose, the number of rootson calli decreased significantly when media for either callusmaintenance or plant regeneration were amended with citric acid.EDTA was less effective in reducing the number of roots thancitric acid. An increase in the amount of a 31kDa (P1) polypeptideunder conditions which favoured a reduction in root formationwas observed. Key words: Wheat, Triticum aestivum, IAA-degrading compounds, indoleacetic acid, root number.     

Should we measure serum or plasma lead concentrations?

ProjectSerum samples may not be appropriate to assess lead (Pb) concentrations because they may contain artificially higher Pb concentrations compared with those measured in plasma samples. Here, we compared Pb concentrations in serum versus heparin plasma separated from blood collected with or without vacuum. We have also examined the effects of sample standing time on Pb concentrations measured in serum, heparin plasma, and EDTA plasma.ProcedureWe studied plasma and serum samples from twelve healthy subjects. Blood samples were collected via venous drainage phlebotomy with and without vacuum into trace metal free tubes containing no anticoagulants (serum), or lithium heparin, or EDTA (to obtain plasma). Variable sample standing times (0, 5, and 30 min) prior to centrifugation were allowed. Plasma and serum Pb and iron concentrations were determined by inductively coupled plasma mass spectrometry. Plasma and serum cell-free hemoglobin concentrations were measured.ResultsPb concentrations in serum and in heparin plasma from blood samples collected with or without vacuum were similar and not associated with significant changes in iron or hemoglobin concentrations. The sample standing time (up to 30 min) did not affect Pb concentrations in serum or in heparin plasma, which were approximately 50% lower than those found in EDTA plasma.ConclusionsSerum or heparin plasma separated from blood samples collected via venous phlebotomy with or without vacuum are appropriate medium to assess Pb concentrations, independently of the sample standing time.