A second-generation blood substitute (perfluorodichlorooctane emulsion) does not activate complement during an ex vivo circulation model of bypass.

OBJECTIVES: To examine whether a second-generation perfluorocarbon (PFC) blood substitute added to the cardiopulmonary bypass (CPB) prime influences complement production. DESIGN: A prospective, randomized, single-blinded, ex vivo model. SETTING: A university hospital, laboratory, and clinics. PARTICIPANTS: Ten healthy adult consented volunteer blood donors (five men, five women). INTERVENTIONS: Ex vivo closed-loop extracorporeal circuit including membrane oxygenator, tubing, and filter primed with crystalloid or crystalloid plus PFC was circulated for 1 hour with the addition of 500 mL of heparinized fresh human whole blood. MEASUREMENTS AND MAIN RESULTS: Laboratory specimens were drawn from the circuit at 10-minute intervals for 1 hour and measured for complement (C3a, Bb fragment) concentrations, blood gases, fibrinogen concentration, platelet count, and hematocrit. In the PFC group, C3a and Bb fragments were equal to or less than those in the group that received crystalloid alone. CONCLUSION: The second-generation PFC added to the prime of a CPB circuit does not independently increase complement production.

Elimination of an artificial immune complex by plasmapheresis in rabbits.

The behaviour of an artificial immune complex was investigated in 15 rabbits. The immune complex was labelled with iodine-125 (125I). The advantage was that the immune complex could not be metabolized without being eliminated by the kidney. This artificial immune complex was injected in different dosages (0.8 micrograms-40 micrograms/rabbit) on 2 consecutive days. One group (n = 9) was treated with plasmapheresis: the other (n = 6) was a control group that did not undergo plasmapheresis. A miniaturized plasmapheresis system eliminated the immune complex very effectively. The increasing concentration of the immune complex in the blood before the next plasmapheresis treatment was probably an expression of mobilization of the immune complex from different organs. The animals of the plasmapheresis group were in better condition than the control group.

The synthesis of a water soluble complement activating polyacrylic acid-IgG polymer.

Polyacrylic acid-IgG polymer (PAA-IgG) activates the classical and alternative pathway of complement as shown by specific Clq-(IgG-PAA) interaction and the generation of C4d, Bb, C3b, C3a and C5a. This water soluble and stable PAA-IgG polymer represents a model substance for the study of humoral and cellular inflammatory mechanisms, mediated by the complement system.

Liquid phase enzyme reactor, a new bioreactor principle.

One of the drawbacks of solid phase enzyme reactors is the sharp decrease in substrate affinity as shown by an increase in the Km app value. To circumvent this problem a liquid phase enzyme reactor system was designed. Biotin labeled L-Tryptophan side chain oxidase (TSO) was directly injected into the plasma circuit of a filter plasmaphoresis system of rabbits and adsorbed onto an Avidin column prior to its reentry into the animal's general blood circulation. In five experiments total L-Tryptophan depletion could be observed in the plasma circuit during 60 minutes of the experiment with complete readsorption of the enzyme to the Avidin column.

Elimination of an artificial immune complex by plasmapheresis in rabbits.

The behavior of an artificial immune complex was investigated in 15 rabbits. The immune complex was labeled with iodine-125 (125I). The advantage was that the immune complex could not be metabolized without being eliminated by the kidney. This artificial immune complex was injected in a dosage of 0.8 microgram, in 2 rabbits, 5.0 micrograms in 4 rabbits, 10 micrograms in 3 rabbits, 20 micrograms in 2 rabbits, and 40 micrograms in 4 rabbits on two consecutive days. One group (n = 9) was treated with plasmapheresis: the other (n = 6) was a control group that did not undergo plasmapheresis. A miniaturized plasmapheresis system eliminated the immune complex very effectively with an average exchange volume of 95.7 ml per treatment. The increasing concentration of the immune complex in the blood before the next plasmapheresis treatment was probably an expression of mobilization of the immune complex from different organs. The animals of the plasmapheresis group were in better condition than the control group.

Consumption of complement and activation of human neutrophils by an artificial immune complex: polyacrylic acid-IgG-polymer.

An artificial immune complex consisting of IgG covalently bound to polyacrylic acid (PAIGP) was prepared and investigated for its influence on a number of immunological reactions attributed to natural immune complexes. PAIGP consumed complement in a fast reaction. Complement consumption was complete after 10 min of incubation of guinea-pig serum with PAIGP. The concentration of PAIGP for 50% consumption was 2.3 micrograms/ml. PAIGP induced a chemiluminescence response in human peripheral polymorphonuclear leukocytes. This response was elicited in the absence and presence of serum and in whole blood. The response was maximal for leukocytes in the absence of serum and rather low in whole blood. The induction of chemiluminescence by PAIGP was inhibited by monoclonal antibodies to one of the Fc receptors of leukocytes (anti-Leu 11B), while unrelated antibodies had no influence on the chemiluminescence induced by PAIGP. PAIGP also stimulated the production of superoxide anion by polymorphonuclear leukocytes. The efficacy of PAIGP in stimulation of superoxide production was comparable to phorbol myristate acetate (PMA) and opsonized zymosan. PAIGP induced the discharge of elastase, a constituent of the azurophile granules of PMN leukocytes. Here, PAIGP was a rather weak stimulus compared to opsonized zymosan. PMA proved unable to induce elastase release. Thus, PAIGP induced a number of biological reactions usually brought about by naturally occurring antigen antibody complexes.

The synthesis of L-tryptophan degrading bioreactors.

To design a bioreactor for removing the potential cancer nutrient L-tryptophan from blood, the L-tryptophan degrading enzyme tryptophan side chain oxidase (TSO) was chemically bound to glutaraldehyde activated gamma amino silane silica and to Zetaffinity microcolumns consisting of a glutaraldehyde activated polyacrylic-cellulose copolymer. Five experiments were carried out in sheep and six experiments in rabbits using a closed circuit plasmapheresis bioreactor system. L-tryptophan in sheep was degraded by the silica bioreactor in a single pass to undetectable levels as measured by high performance liquid chromatography (HPLC). Zetaffinity bioreactors degraded L-tryptophan in rabbits to more than 95% in a single pass. Whole blood L-tryptophan levels changed little throughout the experiment indicating a vast extravascular tryptophan pool. Enzyme leakage from the bioreactor was less than 10(-5) IU TSO per ml plasma. The procedures were tolerated well by the animals without any change in vital signs.

Standardization of methods for measuring plasminogen activator inhibitor activity in human plasma.

We have standardized the measurement of plasminogen activator inhibitor type 1 (PAI-1) activity in plasma. One-chain tissue-type plasminogen activator (t-PA; EC 3.4.21.31; final activity, 5 int. units/mL) was incubated with plasma (final dilutions 1:4 to 1:40) in phosphate buffer (pH 7.4, ionic strength = 0.15) for 15 min at 37 degrees C, followed by acidification and measurement of residual t-PA activity by an amidolytic method. The PAI-1 activity assay was 98% specific for PAI-1 activity in samples from both pregnancy and nonpregnancy, and varied linearly with added plasma volume when the percent inhibition of t-PA was between 8% and 50%. For the standardized method, analytical recovery was 93 +/- 5%, the detection limit was 1.6 arbitrary units per milliliter (1 arb. unit of PAI-1 activity = inhibition of 1 int. unit of t-PA activity), and total imprecision was 10.2 (SD 0.7) arb. units/mL (CV = 7%, n = 20). The average PAI-1 activity in 10 healthy individuals drawn between 0800 and 1000 hours was 23.9 +/- 15.4 arb. units/mL. Compared with the standardized assay, two of three previously described assays underestimated PAI-1 activity in plasma by 77% and 85%, respectively.

Optimum conditions for the stabilization and measurement of tissue plasminogen activator activity in human plasma.

Current studies show a more than 50-fold variation in the estimated level of tissue-type plasminogen activator (t-PA) activity in normal resting blood samples that result from major differences in the methods used to sample, preserve, and assay t-PA activity in blood. In this study we developed optimized methods for stabilizing and measuring t-PA activity in plasma by using a coupled plasminogen-chromogenic substrate (amidolytic) assay. To maximize the recovery of t-PA activity, blood should be acidified within 60 seconds after being drawn by adding 2 parts whole blood to 1 part 0.5 mol/L sodium acetate, pH 4.2. This method prevents hemolysis and eliminates 70% of the alpha 2-plasmin inhibitor. Optimum conditions for measuring t-PA activity are pH 8.0 to 8.3 (37 degrees C), ionic strength 0.02 to 0.04, 0.5 mumol/L plasminogen, 80 micrograms/ml CNBr-cleaved fibrinogen, and a chromogenic substrate concentration of 0.65 mmol/L D-valyl-leucyl-lysyl-p-nitroanilide, 0.25 mmol/L D-valyl-phenylalanyl-lysyl-p-nitroanilide, or 0.2 mmol/L D-norleucyl-hexahydrotyrosyl-lysyl-p-nitroanilide. The final assay is linear with respect to added one-chain t-PA, two-chain t-PA, and acidified plasma. There was no difference in t-PA activity measured with ethylenediaminetetraacetic acid anticoagulant versus that measured with citrate anticoagulant after correction for dilution effects (average resting t-PA activity in plasma from 20 healthy individuals = 1.59 IU/ml). We conclude that assay conditions can have major effects on the measurement of t-PA activity in plasma and that suboptimal conditions may result in a significant underestimation of t-PA activity.

Diurnal variation of tissue-type plasminogen activator and its rapid inhibitor (PAI-1).

Previous studies have shown that overall fibrinolytic activity in blood follows a diurnal rhythm with a peak in the morning and a trough in the evening. The purpose of this study was to determine which fibrinolytic factor(s) was responsible for this diurnal rhythm. Resting and postvenous occlusion tissue-type plasminogen activator (t-PA) activity, resting t-PA antigen, and resting plasminogen activator inhibitor 1 (PAI-1) activity were measured in the morning and evening in 33 healthy men (mean age, 31 years) and in 15 patients (mean age, 57 years) with previous myocardial infarction or unstable angina. PAI-1 activity and t-PA antigen were significantly higher (p less than 0.01) in the morning compared with the evening in controls and patients. In contrast, resting t-PA activity was significantly lower in the morning (p less than 0.01) in both groups and was inversely correlated with PAI-1 activity (r = -0.57, p less than 0.0001). Postvenous occlusion t-PA activity and t-PA capacity were not significantly different between morning and evening in either group. Because t-PA antigen levels and PAI-1 activity were highest in the morning, the variation in t-PA activity was probably not due to decreased secretion of t-PA but instead to changes in the secretion of PAI-1. Our findings indicate that diurnal variations in PAI-1 activity may reduce fibrinolytic activity in the morning in healthy individuals and in patients with coronary artery disease.

Successful long-term use of a miniaturized plasmapheresis circuit in rabbits.

Dual lumen silicon rubber right atrial catheters were implanted into the jugular of 8 rabbits and tunneled subcutaneously to exit sites between the ears. Using a miniaturized tubing-pump system, blood flow rates of 25 ml/min could be achieved for up to 3 1/2 hours without sign of hemolysis in an extracorporeal blood circuit. Seven catheters functioned an average of 75 +/- SE 19 days (range 24-117). One catheter remains functional after 176 days. Infection and thrombosis were the main reasons for failure. 20 plasmaphoresis experiments were carried out in four heparinized rabbits (blood flow rates 15 ml/min, plasmaflux 1.5-2.0 ml/min) using polypropylene minifilters (average pore size, 0.55 micron) with the plasma recirculated back into the animal. No hemolysis was detectable throughout the 4 hr experiment. Plasma proteins with a MW of 69 X 10(3) to 3 X 10(6) (Albumin, LDH, SGOT, SGPT, CPK, fibrinogen, LDL) showed a sieving coefficient close to 1.0. The good filtration performance and the absence of side effects make this system a possible use for plasmaphoresis in neonates.

Estimation of surface-bound heparin activity: a comparison of methods.

We compared two assays for estimating the amount of active heparin bound to a catheter surface: 1) a kinetic assay based on the inactivation of thrombin by antithrombin III, and 2) thrombin uptake. Both assays were used to estimate the amount of heparin activity on a series of catheters coated with no heparin, covalently bound heparin, and ionically bound heparin. The kinetic assay produced estimates of surface-bound heparin activity and showed that some binding methods resulted in destruction of most of the heparin's biologic activity. In contrast, the thrombin uptake assay did not correlate with the amount of heparin activity on the catheter surface. Substantial thrombin uptake was found on surfaces coated with no heparin or inactive heparin, while low thrombin uptake was found on surfaces with high levels of heparin activity in the kinetic assay. We conclude that: 1) a kinetic assay based on the heparin accelerated inactivation of thrombin by antithrombin III can be used to estimate the amount of active heparin bound to a catheter surface, and 2) thrombin uptake studies do not correlate with heparin activity and do not predict which heparin binding method will result in the highest concentration of active heparin on the catheter surface.

Accurate coagulation studies from heparinized radial artery catheters.

We studied the feasibility of obtaining accurate coagulation studies from indwelling, heparinized radial artery catheters in 28 patients after cardiac surgery. PTT assay was chosen because of its frequent clinical use. Thrombin time assay was chosen because it is a component of the coagulation screening panel and because it is useful in assessing heparin contamination of specimens. We conclude that PTT results are reliable when the dwell volume (0.6 ml for the catheter and extension tubing in our study) and an additional 4.5 ml have been discarded (5.1 ml total discard volume). We recommend the collection of samples for thrombin time assay from a separate venous site because results on samples from heparinized arterial catheters are unpredictable.

Evaluation of a new dynamic viscometer for measuring the viscosity of whole blood and plasma.

We evaluated a new type of dynamic viscometer, the Sonoclot Coagulation Analyzer, for use in measuring the viscosity of whole-blood and plasma. Such information can be useful in monitoring patients with hyperviscosity syndromes, e.g., from multiple myeloma. A vibrating Teflon or plastic probe continuously measures dynamic viscosity. The instrument can be calibrated to measure a range of viscosities from 0.69 to 23 cP (mN X s X m-2) or more. The coefficient of variation at 0.69 cP was 3-4% for measurements with the Teflon probe, 7-9% with the plastic probe. Viscosity measured at 37 degrees C for plasma and whole-blood samples from 20 normal patients was 1.22 (SD 0.05) cP and 3.63 (SD 0.52) cP, respectively. Dynamic viscosity measured in blood samples from a single source, with contrived hematocrits ranging from 0 to 89%, increased exponentially as a function of hematocrit, confirming previous studies. Overall, we found this instrument simple and quick to operate, producing accurate, precise viscosity measurements over at least a 40-fold range of viscosity.

Hemodynamic measurements in a sheep model with a hollow fiber artificial kidney containing modified cellulose.

HFAK-RC caused pronounced leukopenia, increase in TXB2 levels in plasma and hemodynamic pressure changes as a reflection of complement activation during EC in sheep. In contrast no increase in TXB2 levels and no changes in hemodynamics are observed with HFAK-MC. The leukopenia and granulocytopenia in the latter is much less pronounced and probably reflects the phenomenon "frustrated phagocytosis".