Effect of blood collection tube types on the measurement of human epidermal growth factor

We observed significant differences in measured human epidermal growth factor (hEGF) levels for the same individual's serum/plasma samples between different tube types (glass, polystyrene, plastic with clot activator, plastic without clot activator, plastic with EDTA, polypropylene tubes). For all individuals, hEGF levels in plasma were found to be below the detection limit. The discrepancy of the hEGF levels in serum and plasma was attributed to the platelet derived EGF by analyzing platelet lyzate with size exclusion chromotography and demonstrating the immunoreactivity of the fractions corresponding to the pre-proEGF and/or proEGF elution time. Besides, samples of females showed much higher EGF levels than those of males in certain test tube types. As a conclusion, all blood samples should be taken and stored in the same type of test tubes in order to make precise measurements for hEGF. And, the measured hEGF level in blood is susceptible to changes with blood clotting.     

Hemocompatibility evaluation of poly(glycerol-sebacate) in vitro for vascular tissue engineering

Poly(glycerol-sebacate) (PGS) is an elastomeric biodegradable polyester that could potentially be used to engineer blood vessels in vivo. However, its blood-material interactions are unknown. The objectives of this study were to: (a) fabricate PGS-based biphasic tubular scaffolds and (b) assess the blood compatibility of PGS in vitro in order to get some insight into its potential use in vivo. PGS was incorporated into biphasic scaffolds by dip-coating glass rods with PGS pre-polymer. The thrombogenicity (platelet adhesion and aggregation) and inflammatory potential (IL-1beta and TNFalpha expression) of PGS were evaluated using fresh human blood and a human monocyte cell line (THP-1). The activation of the clotting system was assessed via measurement of tissue factor expression on THP-1 cells, plasma recalcification times, and whole blood clotting times. Glass, tissue culture plastic (TCP), poly(l-lactide-co-glycolide) (PLGA), and expanded polytetrafluorethylene (ePTFE) were used as reference materials. Biphasic scaffolds with PGS as the blood-contacting surface were successfully fabricated. Relative to glass (100%), platelet attachment on ePTFE, PLGA and PGS was 61%, 100%, and 28%, respectively. PGS elicited a significantly lower release of IL-1beta and TNFalpha from THP-1 cells than ePTFE and PLGA. Similarly, relative to all reference materials, tissue factor expression by THP-1 cells was decreased when exposed to PGS. Plasma recalcification and whole blood clotting profiles of PGS were comparable to or better than those of the reference polymers tested.     

Calcium redistribution,calcification and stone formation in the parotid gland during experimental stimulation and hypercalcaemia

Summary Distribution and redistribution of intra- and pericellular calcium was investigated in the parotid gland of rats under secretory stimulation and hypercalcaemia. The effects of hypercalcaemia and secretory stimulation and of the combination of both were compared. Calcium content was determined by atomic absorption spectrometry. Calcium distribution within the tissue was demonstrated by light microscopical (GBHA) staining and electron microscopical (pyroantimonate method) cytochemistry in combination with X-ray microanalysis. Typical calcium depot sites were the basal and cellular membranes, the calcium buffer organelles (i.e. mitochondria) the secretory granules and the acinar lumina. After stimulation (by isoprenalin) a decrease of calcium-enriched secretory granules and a depletion of intracellular calcium buffer organelles occurred. During hypercalcaemia (induced by dihydrotachysterol), a calcium overloading of the cell membrane and intracellular buffer organelles without calcification was observed. Combined stimulation and hypercalcaemia induced an excessive calcium overloading of all intra-and extracellular calcium depots with excessive calcium release into the acinar lumina resulting in calcium phosphate aggregates and stone formation. Secretory stimulation and simultaneous hypercalcaemia exert potentiating effects on intracellular and intraluminal calcification proposing an importance for pathogenesis of human sialolithiasis.Supported by Sonderforschungsbereich 34 Endokrinologie     

mPEG表面修饰的PLGA嵌段共聚物的血液相容性评价

本实验室设计合成了三种不同LA／GA比例的mPEG修饰PLGA（PELGA，含15％mPEG），为了评价它们的血液相容性，我们以硅化玻璃试管为阴性对照，未硅化的试管为阳性对照，参照国际标准（ISO10993）和《中华人民共和国国家标准GB／T16886医疗器械生物学评价》方法进行了体外评价实验。试验包括溶血率实验，血小板黏附实验，动态凝血时间实验，凝血时间实验，血浆复钙时间实验和凝血酶原时间实验等综合评价指标。结果表明，合成材料具有优良的血液相容性，材料制成的纳米粒有望应用于静脉注射。     

Blood pressure is lowered by vitamin D (alphacalcidol) during long-term treatment of patients with intermittent hypercalcaemia. A double-blind, placebo-controlled study

There is epidemiologic evidence of a relationship between calcium deficiency and hypertension. The present study evaluated the effects of alphacalcidol, a synthetic analogue of active vitamin D, given to 29 patients with marginal, intermittent hypercalcaemia. Before therapy there was an inverse relationship between serum calcium levels and diastolic blood pressure (p less than 0.02). Treatment with 1 microgram alphacalcidol raised the serum calcium by 0.07 mmol/l during a 6-month, double-blind, placebo-controlled trial and caused a significant reduction of diastolic blood pressure by 9.2 mmHg compared with placebo (p less than 0.01). The study extends previous observations, in normocalcaemic subjects, of inverse relationships between serum calcium and blood pressure indicating a primary disturbance of calcium homeostasis in hypertension. The observation that a physiologic amount of active vitamin D has hypotensive effects agrees with such a concept and suggests a new principle for the treatment of hypertension.     

Plasma-deposited tetraglyme surfaces greatly reduce total blood protein adsorption, contact activation, platelet adhesion, platelet procoagulant activity, and in vitro thrombus deposition

The ability of tetraethylene glycol dimethyl ether (tetraglyme) plasma deposited coatings exhibiting ultralow fibrinogen adsorption to reduce blood activation was studied with six in vitro methods, namely fibrinogen and von Willebrand's factor adsorption, total protein adsorption, clotting time in recalcified plasma, platelet adhesion and procoagulant activity, and whole blood thrombosis in a disturbed flow catheter model. Surface plasmon resonance results showed that tetraglyme surfaces strongly resisted the adsorption of all proteins from human plasma. The clotting time in the presence of tetraglyme surfaces was lengthened compared with controls, indicating a lower activation of the intrinsic coagulation cascade. Platelet adhesion and thrombin generation by adherent platelets were greatly reduced on tetraglyme-coated materials, compared with uncoated and Biospan-coated glass slides. In the in vitro disturbed blood flow model, tetraglyme plasma coated catheters had 50% less thrombus than did the uncoated catheters. Tetraglyme-coated materials thus had greatly reduced blood interactions as measured with all six methods. The improved blood compatibility of plasma-deposited tetraglyme is thus not only due to their reduced platelet adhesion and activation, but also to a generalized reduction in blood interactions.     

Proliferation, differentiation, and calcification of preosteoblast-like MC3T3-E1 cells cultured onto noncrystalline calcium phosphate glass

The purpose of this study was to investigate calcium phosphate glass as a potential biomaterial for hard tissue repair. We prepared calcium phosphate glass using the system CaO-CaF(2)-P(2)O(5)-MgO-ZnO and cultured MC3T3-E1 cells onto the glass in alpha-MEM with beta-glycerophosphatase and ascorbic acid. Proliferation of the cells was determined to evaluate the biocompatibility of the prepared calcium phosphate glass. The alkaline phosphatase activity was measured to examine the osteoblast differentiation. Mineralization was evaluated by staining the calcium precipitates with Alizarin red. Culture onto the calcium phosphate glass exhibited no significant influence on cell proliferation compared to the polystyrene chosen as a control in this experiment (p > 0.05). The alkaline phosphatase activity in the experimental group, however, was enhanced by the calcium phosphate glass significantly at 10-18 days after incubation than that of the control group (p < 0.05). The promotion of bone-like tissue formation by the calcium phosphate glass was observed after 7 days and thereafter. The results of the present study indicate that the prepared calcium phosphate glass affects osteogenesis by increasing calcification of the extracellular matrix.     

Effects of acute hypercalcaemia on blood pressure in subjects with and without parathyroid hormone secretion

AIM: Acute hypercalcaemia increases the blood pressure, but the mechanism is uncertain. It may partly be the result of the concomitant fall in parathyroid hormone (PTH) secretion as PTH has been reported to have a vasodilator effect. To elucidate this, we infused calcium intravenously in subjects with and without PTH secretion. METHODS: Seven thyroparathyroidectomized subjects with undetectable PTH levels and 10 controls were studied twice, once with a calcium clamp technique that increased plasma ionized calcium in two steps of 0.1 mmol L(-1), each step lasting 60 min, and once with a placebo infusion. RESULTS: On the placebo day, blood pressure and all other variables were unaffected in both groups. On the calcium day, systolic blood pressure increased gradually and significantly from end of baseline till end of the calcium infusion in the controls (123.5 +/- 19.8 and 134.2 +/- 17.6 mmHg, P < 0.004) but not in the thyroparathyroidectomized subjects (124.9 +/- 15.7 and 126.0 +/- 20.6 mmHg, P = ns). Serum PTH levels fell promptly in the controls, and in both groups there was a significant increase in serum phosphate. The diastolic blood pressure and pulse rate, and the plasma adrenaline and noradrenaline, plasma renin activity, and serum aldosterone levels were unaffected by the calcium infusion. CONCLUSION: During acute hypercalcaemia the blood pressure increase appears unrelated to catecholamine secretion and the renin-aldosterone system, whereas the fall in PTH secretion may play a contributory role.     

The effect of TiO2 nanotubes in the enhancement of blood clotting for the control of hemorrhage

The main biological purpose of blood coagulation is formation of an obstacle to prevent blood loss of hydraulic strength sufficient to withstand the blood pressure. The ability to rapidly stem hemorrhage in trauma patients significantly impacts their chances of survival, and hence is a subject of ongoing interest in the medical community. Herein, we report on the effect of biocompatible TiO2 nanotubes on the clotting kinetics of whole blood. TiO2 nanotubes 10 microm long were prepared by anodization of titanium in an electrolyte comprised of dimethyl sulfoxide and HF, then dispersed by sonication. Compared to pure blood, blood containing dispersed TiO2 nanotubes and blood in contact with gauze pads surface-decorated with nanotubes demonstrated significantly stronger clot formation at reduced clotting times. Similar experiments using nanocrystalline TiO2 nanoparticles showed comparatively weaker clot strengths and increased clotting times. The TiO2 nanotubes appear to act as a scaffold, facilitating fibrin formation. Our results suggest that application of a TiO2 nanotube functionalized bandage could be used to help stem or stop hemorrhage.     

Value of assessing parathyroid hormone-like activity in a case of extreme hypercalcaemia.

A previously well 70 year old woman was admitted to hospital following a three day history of vomiting and confusion. Her serum calcium was 6.58 mmol/l, phosphate 1.09 mmol/l, and alkaline phosphatase 91 iu/l. The mechanism of this hypercalcaemia was not obvious as there was no evidence of a primary malignancy, lymphadenopathy or hepatosplenomegaly. The calculation of indices of urinary excretion of calcium and phosphate suggested the presence of excessive parathyroid hormone (PTH) activity as the mechanism of hypercalcaemia. Plasma intact PTH, 25-hydroxycholecalciferol, and 1,25-dihydroxycholecalciferol were not raised suggesting the presence of PTH related peptide (rP). This led to a systematic search for a malignancy, which revealed the presence of a high grade B cell non-Hodgkin's lymphoma confined to the bone marrow. Plasma PTH-rP was subsequently shown to be raised confirming the interpretation of the initial urinary and calcium excretion indices. This case highlights the value of standard laboratory measurements such as urinary calcium and phosphate excretion in cases of hypercalcaemia of obscure aetiology, which can complement measurements of PTH and other calcitropic hormones.     

Poly(O-acyl hydroxy L-proline) (II) wetting characteristics and blood clotting on surfaces of poly O-acetyl, butyryl, hexanoyl, dodecanoyl, and benzoyl hydroxy L-proline

Poly O-acetyl, butyryl, hexanoyl, dodecanoyl, and benzoyl hydroxy L-proline (poly[O-acyl Hyp]s) were evaluated as materials for blood contact by means of contact angle and blood clotting time measurements. Critical surface tensions obtained from Zisman plots for all materials were 22-29 dyn/cm, suggesting that these materials may exhibit good blood compatibility. Dispersion and nondispersion force contributions to the surface tension were gamma ds = 1.4, gamma ns = 49.3 dyn/cm, (11.0, 16.2), (19.8, 3.8), (21.2, 4.6) and (14.1, 10.8) for the poly(O-Acetyl, Butyryl, Hexanoyl, Dodecanoyl and Benzoyl Hyp) surfaces, respectively. The materials showed remarkable wetting differences that were dependent on the type of acyl group attached to Hyp. The values of the dispersion and nondispersion components of the surface tension for poly(O-hexanoyl Hyp) and poly(O-dodecanoyl hyp) were very close to those obtained for glutaraldehyde-treated umbilical cord vessels. The blood clotting times on the respective polymer surfaces, obtained by using the kinetic method, were normalized to those of control glass and siliconized glass surfaces. All the poly(O-acyl Hyp)s surfaces showed longer clotting times than those of the poly(L-proline) and glass surfaces. The surfaces of those polymers having longer aliphatic or aromatic acyl groups had longer clotting times than those of the polymers with relatively shorter groups.     

Does ovarian stimulation for in-vitro fertilization induce a hypercoagulable state?

Effects on blood coagulation and fibrinolytic activity during ovarian stimulation for in-vitro fertilization (IVF) were examined in 12 women. Blood samples were taken prior to hormonal stimulation (days 2-3 of the menstrual cycle, mean serum oestradiol concentration 0.16 nmol/l) and the day after ovulation induction with human chorionic gonadotrophin (HCG) (days 10-12, mean serum oestradiol concentration 5.35 nmol/l). We measured whole blood clotting time, whole blood clot lysis time, plasma fibrinogen, factor VII and antithrombin III. The whole blood clotting time was slightly, but not significantly shortened after ovarian stimulation. A significant rise in plasma fibrinogen (P less than 0.001) and reduction in antithrombin III (P less than 0.001) were observed, whereas no change in factor VII was found. The blood fibrinolytic activity was significantly reduced as evaluated by an increase in the clot lysis time (P less than 0.02). These results indicate that ovarian stimulation for IVF may create a state of hypercoagulability.     

Whole blood procoagulant activity in breast and colorectal cancer.

Whole blood procoagulant activity was determined by measuring the recalcification time of citrated blood, with and without the addition of bacterial endotoxin, in patients with breast cancer (n = 39), colorectal cancer (n = 20), benign breast disease (n = 15), benign colorectal disease (n = 11), normal volunteers (n = 15) and inpatients with non-malignant disease (n = 22). The median clotting times of those samples incubated with endotoxin were significantly shorter in the patients with breast and colorectal cancer compared with normal controls. Furthermore, significant differences between the median clotting times of stimulated and unstimulated samples within each subject group were observed only in the two cancer groups. There was no correlation between whole blood procoagulant activity and absolute monocyte counts, with histological staging or with plasma concentrations of plasma fibrinopeptide A. The results suggest that blood from patients with cancer is more sensitive to endotoxin stimulation than that from normal or benign controls, but that in its present form the technique cannot be used to distinguish between malignant and non-malignant disease.     

The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules

Modular tissue-engineered constructs are assembled from sub-mm sized cylindrical collagen gel modules which are covered with a surface layer of human umbilical vein endothelial cells (HUVEC). The resulting construct is permeated by a network of interconnected endothelial cell lined channels to facilitate blood perfusion and nutrient delivery. This design strategy relies critically on the endothelial cells' layer behaving in a non-thrombogenic manner on the module surface and the objective here was to characterize this thrombogenicity. HUVEC prolonged clotting times in whole blood-module mixtures, and enabled slightly heparinized whole blood perfusion of an assembled modular construct in vitro with no increase in platelet loss compared to background levels. Flow cytometry and scanning electron microscopy indicated that HUVEC seeded modules reduced platelet activation and deposition but not leukocyte activation, compared to collagen only modules. Plasma recalcification times on non-stimulated HUVEC were longer compared to stimulated HUVEC but not different than that on collagen only module films and were not prolonged by incubation with a tissue factor blocking antibody. Together these data suggest that a functional non-thrombogenic layer of EC was generated on the module surface and that this layer should be sufficient to maintain continuous blood flow through an engineered modular tissue. In/ex vivo studies are warranted to confirm this conclusion.     

Measurement of the rate of thrombin production in human plasma in contact with different materials.

Thrombin production in plasma in contact with various materials was consistent with a first-order autocatalytic model (d[T]/dt = kp[T]; [T] = thrombin concentration, t = time, kp = thrombin production rate constant) since the initial portion of a semilogarithmic plot of thrombin concentration against time was linear. Thrombin concentration was measured in clotting plasma (phospholipid enhanced or platelet-rich plasma) using a fluorogenic substrate (BMCA) by aliquot sampling at various intervals or more conveniently by monitoring cumulative fluorescence. The latter was generated by the action, on BMCA incubated in the clotting plasma, of the thrombin as it was generated. The thrombin concentration was determined from the first derivative of the S-shaped cumulative fluorescence curve. kp was greater for glass (7.92 x 10(-3) cm/s) than for the other materials (polypropylene, polystyrene, polyethylene and PVA; kp approximately 3.1 x 10(-3) cm/s) in plasma with cephalin without flow. A kp for heparin-PVA could not be determined since the thrombin concentration was too low to be quantified. A larger difference between polyethylene and PVA was noted with platelet-rich plasma without flow while lower values (1.0 x 10(-3) cm/s) were noted in a flow system but at a higher surface to volume ratio. The first-order rate constant can be used in simple models relating production of thrombin at a wall of a tube to its mass transfer away from the wall in flowing blood. One such model predicts that the concentration of thrombin at the wall should become infinite at the point in the tube when the mass transfer coefficient equals kp. According to this model, kp on the order of 10(-4) cm/s would be a useful target for a nonthrombogenic material.     

Microcarrier culture of hepatocytes in whole plasma for use in liver support bioreactors.

Contact activation of plasma clotting may limit the use of some microcarrier types for hepatocyte attachment in a liver assist device. Activation by seven microcarrier types was studied in plasma containing 2-500 units heparin/mL. Clotting was activated by dextran (Cytodex 1 and 2) and collagen-coated (Cytodex 3) microcarriers at 2-25 units/mL (Cytodex 1) and 2-100 units/mL (Cytodex 2 and 3). There was no activation by polystyrene, gelatin, glass or fibronectin-coated polystyrene microcarriers. Compared with culture medium, incubation of HepG2 cells in plasma did not affect cell viability but increased cell number (56.4 versus 65.1 x 10(4) cells; P less than 0.05) and incorporation of [3H]-amino acids into protein (204913 versus 279624 dpm; P less than 0.05). Polystyrene-attached cells demonstrated time-linear protein synthesis, glucose and 7-ethoxycoumarin metabolism. We conclude that polystyrene-attached hepatocytes maintain viability and metabolic activity in plasma and are of potential use in a liver support bioreactor.     

Evaluation of the SonoClot Analyzer for the measurement of platelet function in whole blood.

A viscoelastometer, the SonoClot Coagulation Analyzer, has been proposed for use in the evaluation of platelet function. The purpose of this study was to evaluate systematically this instrument when used with whole blood. Under laboratory conditions, the coefficient of variation (cv) of determinations of whole blood activated clotting time (ACT) on the instrument was approximately 7.0 percent. In contrast, the cv of measurements on whole blood related to the graphic events associated with clot formation ranged from 9.2 to 41.7 percent. Because of the large and variable cvs associated with these measurements of clotting, the SonoClot Analyzer cannot presently be recommended for use in studies designed to examine quantitatively the clotting function in whole blood.     

Plasma calcium fractions and the protein-binding of calcium in normal subjects and in patients with hypercalcaemia and hypocalcaemia

A study is reported of the estimation of plasma calcium fractions and the calcium-binding affinity of plasma proteins in a total sample of 59 people, which included 29 normal subjects and 30 patients with either hypercalcaemia or hypocalcaemia. It was demonstrated that when the sample was considered as a whole there was a significant correlation between the total plasma calcium concentration and the ultrafiltrable, ionized, and protein-bound calcium fractions and between the ultrafiltrable and ionized fractions. We have also demonstrated that in patients with either hypercalcaemia or hypocalcaemia, including acidotic uraemics, the calcium-binding affinity of the plasma proteins did not differ significantly from that in normal subjects. A significant correlation was also found between the total plasma calcium concentration and the ultrafiltrable, ionized and protein-bound calcium fractions when the normal subjects and the groups of patients with hypercalcaemia and hypocalcaemia due to chronic renal failure were considered as separate groups. The group of patients with hypercalcaemia included patients both with hyperparathyroidism and with hypercalcaemia due to other causes.     

Mitigation of coagulation by removing clotting factors part 2: heparin-free extracorporeal circulation in a porcine model

A subpopulation of patients would benefit from an anticoagulation strategy during extracorporeal circulation (ECC) that does not involve systemic administration of heparin and protamine. Inhibition of coagulation by adsorption of plasma clotting factors using protamine immobilized on a Sepharose matrix (PSM) has been explored. This investigation extends previous in vitro studies and demonstrates the feasibility of heparin-free ECC. In a porcine ex vivo circuit, plasma was separated from blood via plasmapheresis, passed through a column containing PSM beads, and returned to the animal. Hemodialyzers and stents were placed in the circuit before, during, and after ECC and examined for device thrombosis. After 90 minutes, prothrombin time (PT) was prolonged >10 times the baseline, and blood clotting Factors I, II, VIII, and X were decreased significantly (>90%); this state was maintained for 2.5 hours without detectable adverse consequences. After cessation of ECC, PT approached normal levels within 60 minutes. Examination of hemodialyzers and coronary stents placed in the circuit revealed that the removal of clotting factors significantly reduced device thrombosis and that transfusion of homologous blood ( approximately 10% V/V) resulted in immediate restoration of hemostasis. It is possible to remove clotting factors from circulating blood to allow extracorporeal circulation of blood without the use of heparin.     

Polyhemoglobin-fibrinogen: a novel oxygen carrier with platelet-like properties in a hemodiluted setting

Polyhemoglobin (polyHb) is currently being assessed in phase III trials under various formulations. At present, none contain clotting factors or platelet substitutes to aid in hemostasis. We have prepared a novel blood substitute that is an oxygen carrier with platelet-like activity. This is formed by crosslinking fibrinogen to hemoglobin to form polyhemoglobin-fibrinogen (polyHb-Fg). This was studied and compared to polyHb for its effect on coagulation both in vitro and in vivo. In the in vitro experiments, PolyHb-Fg showed similar clotting times as whole blood, whereas polyHb showed significantly higher clotting times. This result was confirmed in in vivo experiments using an exchange transfusion rat-model. Using PolyHb, exchange transfusion of 80% or more increased the normal clotting time (1-2 mins) to > 10 mins. Partial clots formed with PolyHb did not adhere to the tubing wall. With PolyHb-Fg, a normal clotting time is maintained, even with 98% exchange transfusion.