Water-soluble polymers such as dextran and polyethylene glycol are known to induce aggregation and size growth of phospholipid vesicles. The present study addresses the dependence of these processes on vesicle size and concentration, polymer molecular weight, temperature, and compartmentalization of the vesicles and polymers, using static and dynamic light scattering. Increasing the molecular weight of the polymers resulted in a reduction of the concentration of polymer needed for induction of aggregation of small unilamellar vesicles. The aggregation was fully reversible (by dilution), within a few seconds, up to a polymer concentration of at least 20 wt %. At relatively low phosphatidylcholine (PC) concentrations (up to approximately 1 mM), increasing the PC concentration resulted in faster kinetics of aggregation and reduced the threshold concentration of polymer required for rapid aggregation (CA). At higher PC concentrations, CA was only slightly dependent on the concentration of PC and was approximately equal to the overlapping concentration of the polymer (C*). The extent of aggregation was similar at 37 and 4 degrees C. Aggregation of large unilamellar vesicles required a lower polymer concentration, probably because aggregation occurs in a secondary minimum (without surface contact). In contrast to experiments in which the polymers were added directly to the vesicles, dialysis of the vesicles against polymer-containing solutions did not induce aggregation. Based on this result, it appears that exclusion of polymer from the hydration sphere of vesicles and the consequent depletion of polymer molecules from clusters of aggregated vesicles play the central role in the induction of reversible vesicle aggregation. The results of all the other experiments are consistent with this conclusion.

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The state of aggregation and the steady-state size of mixed aggregates made of phospholipids and surfactants are both determined by the surfactant/lipid ratio in the mixed aggregates (Re). Water-soluble polymers, such as dextrans and polyethylene glycols (PEGs) of different molecular weights, induce reversible aggregation of phospholipid vesicles, mostly due to dehydration of the vesicle surface and depletion forces, and only at much higher concentrations, PEGs (but not dextran) also induce irreversible size growth of the vesicles. Here we show that the water-soluble polymers dextrans and PEGs do not affect the vesicle-micelle phase boundaries in mixtures of phosphatidylcholine and the anionic surfactant sodium cholate. By contrast, these polymers affect markedly the steady-state size of cholate-containing vesicles. As compared with pure phosphatidylcholine vesicles, the cholate-containing vesicles have a lower tendency to undergo polymer-induced aggregation, probably due to the electrostatic repulsion between the negatively charged vesicles, but a higher tendency to undergo irreversible size growth at relatively low polymer concentrations. Such irreversible size growth was observed not only for PEG but also for dextran, which in the absence of cholate is incapable of inducing vesicle size growth. These findings are consistent with the prevailing concept that the polymer-induced size growth is due to the effect of large structural fluctuations in the bilayers of deformed aggregated vesicles, the surface of which is dehydrated by the polymer. The presence of cholate in the bilayers at sufficiently high concentrations induces such fluctuations, yielding irreversible size growth within the clusters of dehydrated vesicles formed upon mixing with polymers.

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Dilution of phospholipid-detergent mixed micellar systems results in an increase of the fraction of monomeric detergent and, consequently, in a decrease of the effective ratio Re between non-monomeric detergent and phospholipid. The value of Re has been previously shown to be the main determinant of the state of aggregation in such mixtures: at Re values below a critical value ReSAT, the mixture is vesicular; at Re values higher than ReSOL, the mixture is micellar, whereas within the range of ReSAT-ReSOL, vesicles and micelles co-exist. Albumin binds bile salts. Therefore, in PC-cholate mixtures, Re is reduced by the presence of albumin in the system. Within the range of PC concentrations of 2-23 mM, cholate concentrations of 2-15 mM and BSA concentrations of 0-100 mg/ml, binding of cholate to BSA results in reduction of the effective cholate concentration to the extent of 0.11 mM cholate per 1 mg/ml BSA, namely up to 7 cholate molecules bind to each BSA molecule. Yet, the values of ReSAT and ReSOL are essentially independent of BSA. In addition, at any given Re value, the size of vesicles made by dilution of mixed micelles is a complex function of albumin and PC concentrations. Possible mechanistic details which may cause this effect are discussed. These effects of albumin on the state of aggregation of PC-cholate mixtures must be taken into account in studies of such mixtures in the presence of albumin when other effects of albumin (e.g., on phospholipolysis) are investigated. Practical conclusions are reached with respect to the procedures that can be used to prepare vesicles of identical composition and size in the presence of different concentrations of albumin.

B-CLL, the most common type of leukemia in adults, is heterogeneous with a varying clinical course ranging from indolent to aggressive disease. CLL patients with mutated Ig V(H) genes have a good prognosis while those with non-mutated Ig V(H) status and expression of the ZAP70 protein tyrosine kinase generally have aggressive disease and shorter survival. Testing for IgV(H) mutations is not routinely performed due to cost and ZAP70 testing is still technically difficult. As these tests remain generally inappropriate for B-CLL patient screening, we tested the capacity of the new Beckmann Coulter LH750 blood analyzers to provide additional morphometric information on the CLL cell population. These analyzers use a highly informative positional parameter technology - VCS (Volume, Conductivity, Scatter) technology, and automated RPD analysis which provide the Mean and Standard Deviation of Volume, Conductivity and Scatter (VCS) for each of the main WBC types (neutrophils, lymphocytes, monocytes and eosinophils), thereby enabling the detection of abnormal WBC populations associated with various hematological and non-hematological disorders. We attempted to determine if the RPD generated could differentiate between the ZAP70pos and ZAP70neg B-CLL subgroups as determined by high resolution quantitative ZAP70 flow cytometry analysis (1). This study included 30 newly diagnosed untreated CLL patients. CBC was performed on all samples using automated leukocyte differentials in the Beckmann-Coulter LH750. Statistical analysis was performed with Medcalc® 8.1.1.0. Results show that there was no correlation between the WBC number, ZAP70pos expression (found in 59% of the cases) and lymphocyte volume, scatter, or conductivity or SD of these parameters. However, differences in the automated morphology of the Zap70neg compared with Zap70pos CLL lymphocytes was detected. When compared to normal lymphocyte volume (85.8±14.5), the Lymphocyte Mean Volume (MLYV) was lower in the ZAP70neg samples (77.7±17.6) (p<0.05), while the ZAP70pos samples had increased volumes (87.5±15.8), similar to values reported in other myeloproliferative states. The conductivity of the ZAP70neg cells was reduced (110±15.0) below normal (117±11.5) (p=0.038), while the ZAP70pos cells had conductivity similar to the control group. Higher levels of lymphocyte apoptosis were clearly observed in the ZAP70neg samples by VCS. When using ROC (receiver operating curve analysis) to detect the potential ZAP70neg cases, the cut off proposed is MLYV < 76 with a sensitivity of 69.2% and a specificity of 70% AUC 0,650. Herein we propose an entirely new approach which may provide a novel diagnostic screening approach to enable identification of CLL patients who do not require further complex testing. This data is derived directly from the CBC of the Beckman-Coulter LH750 analyzers at no extra cost. As it will be necessary to analyze many more cases from different laboratories, an international working group has been formed in an attempt to confirm these early observations.