Edward F. Leonard

BLOOD ELEMENTS AT FOREIGN SURFACES: A BIOCHEMICAL APPROACH TO THE STUDY OF THE ADSORPTION OF PLASMA PROTEINS*

When a foreign surface, solid, liquid, or gas, is brought into contact with a protein solution, a certain amount of the dissolved protein will be adsorbed to the surface. This process is consistent with the Gibbs theory of surface energy and may be described by the adsorption isotherms of Freudlich or Langmuir.l The amount of protein adsorbed and the characteristics of the protein monolayer depend mainly on the nature of the foreign surface and the structure and concentration of the proteins in solution. It is impossible in the frame of this presentation to cover the vast amount of data on protein adsorption that has accumulated over the past two decades, but reference will be made to two of several excellent reviews that have been published on this Despite development of sophisticated experimental methods, it is not yet possible to infer, from the behavior of a surface (metal, glass, or polymer) in one system, its response in another. This unpredictability is unfortunate in the context of this conference, because an extrapolation from the behavior of surfaces in vitro to their thrombogenic behavior in vivo would allow prediction of the clinical usefulness of candidate biomaterials. Before a protein molecule can be adsorbed to a surface it must become located at a distance from the surface that will allow interaction between the molecular forces associated with the surface and the protein molecule. This approach is governed by diffusion, but the characteristics of the adsorbing surface also determine the nature of the bond and what changes may take place in the configuration of the bound protein molecule. Protein molecules in aqueous solution are compact structures, globular or fibrillar, depending on characteristics of their polypeptides. The effect of dissolution on the hydrophobic and hydrophilic amino acid residues of a polypeptide chain is reduction of the number of hydrophobic amino acids on its surface and exposure of hydrophilic amino acids. Hydrogen bonds, hydrophobic interactions, ionic and polar forces facilitate such conformational changes, so that the final result is a hydrated, compact protein molecule with an increased number of surface hydrophilic groups, many hydrophobic groups

THE DEPOSITION OF REJECTED MATTER IN MEMBRANE SEPARATION PROCESSES

The mechanism by which particles rejected at a membrane filter that is operated in cross-flow are returned to the feed stream has remained unclear. Comparisons with concentration polarization during molecular rejection raise difficulties because molecular diffusivities are much larger than those considered plausible for particles. In the present work particles are postulated not to return to the bulk but rather to flow along the surface of the membrane in a layer whose thickness increases in the direction of feed flow. A model for both the steady and transient case is solved using the method of characteristics. The role of the rheology of the close-packed layer is emphasized.

Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system

Demand is increasing for therapeutic biopharmaceuticals such as monoclonal antibodies. Achieving maximum production of these recombinant proteins under developmental time constraints has been a recent focus of study. The majority of these drugs are currently produced in altered Chinese hamster ovary (CHO) cells due to the high viability and the high densities achieved by these cells in suspension cultures. However, shortening the process of developing and isolating high-producing cell lines remains a challenge. This article focuses on current expression systems used to produce biopharmaceuticals in CHO cells and current methods being investigated to produce biopharmaceuticals more efficiently. The methods discussed include modified gene amplification methods, modifying vectors to improve expression of the therapeutic gene and improving the method of selecting for high-producing cells. Recent developments that use gene targeting as a method for increasing production are discussed.

Is the Vroman effect of importance in the interaction of blood with artificial materials

The successive displacement of plasma proteins adsorbed to artificial surfaces (biomaterials) is well documented, mostly by specially designed experiments that stretch out the effect in time and space. Analysis of displacement has been focused principally on molecular events on the adsorbing surface. In this paper attention is directed rather to the antecedent transport phenomena necessary to deliver successive proteins to a surface. The different limitations on protein arrival fixed by the total quantity present and by the rates of transport of super-sufficient quantities are distinguished. The transport perspective is then used to ask, and partly answer, the question: Can protein displacement be responsible for patterns of thrombus formation and cellular adhesion that are seen on the blood-wetted surfaces of devices found in medical practice: artificial organs and vascular prostheses? Calculations and a small amount of preliminary data suggest that such patterns may form when blood is introduced into these devices, particularly in the neighborhood of boundary shapes that cause separated flows.

Efficient capture of circulating tumor cells with a novel immunocytochemical microfluidic device

Ability to perform cytogenetic interrogations on circulating tumor cells (CTCs) from the blood of cancer patients is vital for progressing toward targeted, individualized treatments. CTCs are rare compared to normal (bystander) blood cells, found in ratios as low as 1:10(9). The most successful isolation techniques have been immunocytochemical technologies that label CTCs for separation based on unique surface antigens that distinguish them from normal bystander cells. The method discussed here utilizes biotin-tagged antibodies that bind selectively to CTCs. The antibodies are introduced into a suspension of blood cells intending that only CTCs will display surface biotin molecules. Next, the cell suspension is passed through a microfluidic channel that contains about 9000 transverse, streptavidin coated posts. A CTC making contact with a post has the opportunity to engage in a biotin-streptavidin reaction that immobilizes the cell. Bystander blood cells remain in suspension and pass through the channel. The goal of the present study is to establish the technical performance of these channels as a function of antigen density and operating conditions, especially flow rate. At 18 μL/min, over 70% of cells are captured at antigen densities greater than 30 000 sites/cell while 50% of cells are captured at antigen densities greater than 10 000. It is found that lower flow rates lead to decreasing cell capture probabilities, indicating that some streamlines develop which are never close enough to a post to allow cell-post contact. Future modeling and streamline studies using computational fluid dynamics software could aid in optimization of channel performance for capture of rare cells.

A method for the estimation of hydration state during hemodialysis using a calf bioimpedance technique

Although many methods have been utilized to measure degrees of body hydration, and in particular to estimate normal hydration states (dry weight, DW) in hemodialysis (HD) patients, no accurate methods are currently available for clinical use. Biochemcial measurements are not sufficiently precise and vena cava diameter estimation is impractical. Several bioimpedance methods have been suggested to provide information to estimate clinical hydration and nutritional status, such as phase angle measurement and ratio of body fluid compartment volumes to body weight. In this study, we present a calf bioimpedance spectroscopy (cBIS) technique to monitor calf resistance and resistivity continuously during HD. Attainment of DW is defined by two criteria: (1) the primary criterion is flattening of the change in the resistance curve during dialysis so that at DW little further change is observed and (2) normalized resistivity is in the range of observation of healthy subjects. Twenty maintenance HD patients (12 M/8 F) were studied on 220 occasions. After three baseline (BL) measurements, with patients at their DW prescribed on clinical grounds (DW(Clin)), the target post-dialysis weight was gradually decreased in the course of several treatments until the two dry weight criteria outlined above were met (DW(cBIS)). Post-dialysis weight was reduced from 78.3 +/- 28 to 77.1 +/- 27 kg (p < 0.01), normalized resistivity increased from 17.9 +/- 3 to 19.1 +/- 2.3 x 10(-2) Omega m(3) kg(-1) (p < 0.01). The average coefficient of variation (CV) in three repeat measurements of DW(cBIS) was 0.3 +/- 0.2%. The results indicate that cBIS utilizing a dynamic technique continuously during dialysis is an accurate and precise approach to specific end points for the estimation of body hydration status. Since no current techniques have been developed to detect DW as precisely, it is suggested as a standard to be evaluated clinically.

Sorption kinetics of binary protein solutions: General approach to multicomponent systems

Abstract In a previous paper the Langmuir-Hinshelwood (L-H) kinetic approach to modeling adsorption of human γ-globulin (G), on an artificial surface (quartz) was tested against new experimental data. The principal purpose of this paper is to explore the extension of that approach to a binary system as an example of a putative general approach for multicomponent systems. In particular the previous results along with new results from a second single-protein system, human albumin (A), modeled as in the previous study, are combined with new results for the binary system γ-globulin-albumin.

Estimation of normal hydration in dialysis patients using whole body and calf bioimpedance analysis.

Prescription of an appropriate dialysis target weight (dry weight) requires accurate evaluation of the degree of hydration. The aim of this study was to investigate whether a state of normal hydration (DW(cBIS)) as defined by calf bioimpedance spectroscopy (cBIS) and conventional whole body bioimpedance spectroscopy (wBIS) could be characterized in hemodialysis (HD) patients and normal subjects (NS). wBIS and cBIS were performed in 62 NS (33 m/29 f) and 30 HD patients (16 m/14 f) pre- and post-dialysis treatments to measure extracellular resistance and fluid volume (ECV) by the whole body and calf bioimpedance methods. Normalized calf resistivity (ρ(N)(,5)) was defined as resistivity at 5 kHz divided by the body mass index. The ratio of wECV to total body water (wECV/TBW) was calculated. Measurements were made at baseline (BL) and at DW(cBIS) following the progressive reduction of post-HD weight over successive dialysis treatments until the curve of calf extracellular resistance is flattened (stabilization) and the ρ(N)(,5) was in the range of NS. Blood pressures were measured pre- and post-HD treatment. ρ(N)(,5) in males and females differed significantly in NS. In patients, ρ(N)(,5) notably increased with progressive decrease in body weight, and systolic blood pressure significantly decreased pre- and post-HD between BL and DW(cBIS) respectively. Although wECV/TBW decreased between BL and DW(cBIS), the percentage of change in wECV/TBW was significantly less than that in ρ(N)(,5) (-5.21 ± 3.2% versus 28 ± 27%, p < 0.001). This establishes the use of ρ(N)(,5) as a new comparator allowing a clinician to incrementally monitor removal of extracellular fluid from patients over the course of dialysis treatments. The conventional whole body technique using wECV/TBW was less sensitive than the use of ρ(N)(,5) to measure differences in body hydration between BL and DW(cBIS).

Extracellular fluid redistribution during hemodialysis: bioimpedance measurement and model

Intradialytic fluid redistribution may cause hypotension. We hypothesized that measuring extracellular fluid volumes (ECV) with segmental bioimpedance analysis (SBIA) could test a simple, volume-driven model of redistribution among the arm, leg and trunk compartments. Patients (22, 5 females/17 males, with ages 60.2 +/- 9 years, weights 80.7 +/- 15 kg, heights 174 +/- 9 cm) were studied during 30 HD treatments on different days. Hypotensive symptoms (Hypo+) were observed in eight patients. ECVs in the arm, trunk and leg, respectively V(A), V(T) and V(L), were measured at initiation of, and throughout, dialysis. Two variables lambda(A) and lambda(L) were defined as V(A)/V(T) and V(L)/V(T). System dynamics, assuming initial equilibrium, are then described by two rate coefficients k(RL) and k(RA) and two constants beta and gamma. These were obtained using a Marquardt-Levenberg least-squares algorithm. Significant differences (Hypo+ versus Hypo-) for lambda(L) (0.55 +/- 0.13 versus 0.84 +/- 0.3, *p < 0.05) and lambda(A) (0.17 +/- 0.032 versus 0.23 +/- 0.07, **p < 0.01) were found. The small value of lambda(L) might indicate that less leg volume predisposes to hypotension, larger peripheral volume mitigates hypotension. Observed transport ratios indicated that the ratio of limb to trunk volume stabilized during dialysis after an initial adjustment. These data imply encumbered movement of water from the interstitial components around skeletal muscle in the arm and leg to those of the trunk and are useful in predicting anatomical or situational predispositions to hypotension.

The role of convection and diffusion on platelet adhesion and aggregation.

The reactions among clotting components, among platelets, and between these groups are complex biochemical phenomena that importantly affect thrombogenesis, but thrombogenesis is also affected by the physical phenomena of convection and diffusion, which, in turn, are importantly mediated by conditions of flow. The principles that govern convection and diffusion processes are generally well understood, but the significance of these processes in thrombogenesis is in only the early stages of demonstration.